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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.

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.

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.

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 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.

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.

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.

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.

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.

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.

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.

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.

In this enzyme assay for kinetic measurement of alkaline phosphatase (EC 3.1.3.1) activity in human serum by flow injection analysis with electrochemical detection (FIA-EC), the substrate phenyl phosphate is enzymatically hydrolyzed and the phenol produced is quantified by FIA-EC. The standard curve is linear over the range 40 to 500 U/l, and the predicted linearity is up to 1000 U/l. The CV is 5% over the range of the assay. Correlation with the Sclavo Diagnostics nitrophenyl phosphate spectrophotometric technique gave an r of 0.98. This new technique requires as little as 1 μl of serum. The FIA-EC method is not subject to interference by hemolysis, icteria, or lipemia or by electroactive substances such as urate, ascorbate, or acetaminophen. Sensitive measurements free from spectrophotometric interferences are thus possible.

The effect of a perfluorocarbon emulsion oxygen therapeutic (PEOT) on detecting theophylline was explored using a commercial EMIT® (enzyme multiplied immunoassay technique) immunoassay. The EMIT technique is based on a colorimetric reaction, the product of which can be measured spectrophotometrically. The intent was to determine whether the presence of PEOT interferes with this detection. We found that the immunoassay yields excellent quantitative data in the therapeutic range (10–20 ppm theophylline) in 2–10% PEOT, but as the amount of PEOT in the sample increases, the accuracy of the detection method decreases.

Publisher Summary This chapter focuses on the various developments in electrochemical immunoassays and immunosensors after 2002. Electrochemical immunoassay is a solid phase system in which an antibody–antigen reaction takes place but the corresponding electrochemical detection is carried out elsewhere. However, an electrochemical immunosensor is a stand-alone device, with the immunoreaction and electrochemical detection occurring within the same device. There are several strategies for immobilizing a captured antibody on a solid phase, which include covalent attachment, physical adsorption, and electrostatic/physical entrapment in a polymer matrix. The (strept)avidin–biotin interaction technique is used to immobilize various types of biomolecules such as nucleic acids, polysaccharides, and proteins, including the capture antibody in immunoassay/immunosensor systems. Another commonly used affinity-based immobilization technique for capture antibodies in immunoassay systems involves a bacterial antibody-binding protein, the two most common of which are Protein A and Protein G. These proteins bind specifically to antibodies through their nonantigenic (Fc) regions, which allow the antigen binding sites of the immobilized antibody to be oriented away from the solid phase and be available to bind the target analyte. The application of conducting polymers such as polyaniline, polypyrrole, and polythiophene for immobilizing capture antibodies in immunoassay systems is widespread and they may be used in amperometric, potentiometric, and impedimetric immunoassay systems. Self-assembled monolayers (SAMs) are another attractive method for immobilizing the capture antibody in immunoassay systems, which takes advantage of the spontaneous chemisorption of alkanethiols to metals such as gold or silver to assemble highly ordered monolayers. Recently, interdigitated array (IDA) microelectrodes have gained popularity as an alternative transducer in electrochemical immunoassays in which a simple design of an IDA consists of a pair of interdigitated microelectrode fingers.

A sandwich-type heterogeneous enzyme immunoassay with flow-injection analysis for alpha-fetoprotein (AFP) in human serum has been developed. 4-Aminophenol, the product of enzymatic reaction, is detected amperometrically. The interassay CV for this electrochemical enzyme immunoassay was less than 8.2%, with a minimum detection limit for AFP of 0.163 micrograms/L. The calibration curve had a linear range of 0.316-100 micrograms/L. Studies with 48 human maternal serum samples, comparing results by this method with those by a commercial kit, showed a good correlation (r = 0.961). This procedure provides an alternative method for determining low concentrations of AFP in human maternal serum.

The 2001 anthrax alarm in the US raised concerns about the Nation's preparedness to the threat of bioterrorism, and the demand for early warning systems that might be used in the case of a biological attack continues to grow. Here we develop an ultra-sensitive rapid detection method for B. globigii (BG) spores, the simulant of B. anthracis (BA) spores. BG spores were detected by a bead-based sandwich immunoassay with fluorescence detection. Paramagnetic Dynal® beads were used as a solid support, primary antibody was attached to the beads by streptavidin-biotin coupling and the secondary antibody had an alkaline phosphatase (AP) enzyme label. Enzymatic conversion of fluorescein diphosphate (FDP) to fluorescein by AP was measured in real time with λex = 490 nm and λem = 520 nm. The assay was linear from 2.6 × 103–5.6 × 105 BG spores mL−1, and the detection limit was 2.6 × 103 spores mL−1 or 78 spores. All reagent concentrations and incubation times were optimized. The assay time from the moment the spores were introduced to the system was 30 min, and real-time fluorescence detection was done in less than 1 min. Formation of the BG spores–capture beads complex was confirmed by environmental scanning electron microscopy (ESEM). BG spores were detected successfully when doped into Cincinnati tap water to demonstrate the applicability of the developed method to detect the spores in non-buffered media.

Microbead immunoassay with electrochemical detection has been developed as a sensitive and selective technique for rapid and small volume analyses. In this assay, applications of paramagnetic microbeads in a microfluidic system have aided the automation of all assay steps to enable near-continuous monitoring. These mobile microbeads can be transported through microchannels, captured and held at specific points by a magnet. Hence, by performing immunoassay on microbeads, they can be dispersed throughout a small sample of water, where they provide a large surface area to sample volume ratio that enhances the capture of the target antigen by minimizing diffusional distances. They can then be collected magnetically and manipulated to accomplish all the assay steps to determine if any target was captured. In addition, the microbeads can be accommodated in small volumes, which reduces the dilution of the enzyme product in the detection step thus maximizing sensitivity. Further, electrochemical detection coupled with enzyme-labeled immunoassay has led to the development of a sensitive analytical technique. In this area, interdigitated array electrodes are particularly suited to microfluidics. Improved sensitivity is obtained by redox cycling of the species being detected. In this work, the microbead immunoassays is demonstrated for the virus MS2 bacteriophage.

Viruses are one of four classes of biothreat agents, and bacteriophage MS2 has been used as a simulant for biothreat viruses, such as smallpox. A paramagnetic bead-based electrochemical immunoassay has been developed for detecting bacteriophage MS2. The immunoassay sandwich was made by attaching a biotinylated rabbit anti-MS2 IgG to a streptavidin-coated bead, capturing the virus, and then attaching a rabbit anti-MS2 IgG-beta-galactosidase conjugate to another site on the virus. beta-Galactosidase converts p-aminophenyl galactopyranoside (PAPG) to p-aminophenol (PAP). PAPG is electroinactive at the potential at which PAP is oxidized to p-quinone imine (PQI), so the current resulting from the oxidation of PAP to PQI is directly proportional to the concentration of antigen in the sample. The immunoassay was detected with rotating disk electrode (RDE) amperometry and an interdigitated array (IDA) electrode. With an applied potential of +290 mV vs Ag/AgCl and a rotation rate of 3000 rpm, the detection limit was 200 ng/mL MS2 or 3.2 x 10(10) viral particles/mL with RDE amperometry. A trench IDA electrode was incorporated into a poly(dimethyl siloxane) channel, within which beads were collected, incubated with PAPG, and PAP generation was detected. The two working electrodes were held at +290 and -300 mV vs Ag/AgCl, and electrochemical recycling of the PAP/PQI couple by the IDA electrode lowered the limit of detection to 90 ng/mL MS2, or 1.5 x 10(10) MS2 particles/mL.

This research is dedicated to the development of electrode arrays suitable for a more sensitive and faster electrochemical immunoassay system Signal amplification is achieved through coupling the redox cycling of interdigitated array (IDA) electrodes with an enzyme label, /spl beta/-galactosidase. Comb IDAs enhanced the signal 3 times higher than coplanar IDAs. A magnetic bead-based enzyme assay, as a simulation of an immunoassay, has been performed using the comb and coplanar IDAs for detection. Comb IDAs enabled positioning the beads and attached enzyme closer to the sensing electrodes (/spl sim/10 gm). Detection time was less than 1 min. and the limit of detection was 70 amole of /spl beta/-galactosidase.

The objective of this study was to develop a sensitive and miniaturized immunoassay by coupling a microbead-based immunoassay with an interdigitated array (IDA) electrode. An IDA electrode amplifies the signal by recycling an electrochemically redox-reversible molecule. The microfabricated platinum electrodes had 25 pairs of electrodes with 1.6-μm gaps and 2.4-μm widths. An enzyme-labeled sandwich immunoassay on paramagnetic microbeads with mouse IgG as the analyte and β-galactosidase as the enzyme label was used as the model system. β-Galactosidase converted p -aminophenyl β- d -galactopyranoside to p -aminophenol (PAP). This enzyme reaction was measured continuously by positioning the microbeads near the electrode surface with a magnet. Electrochemical recycling occurred with PAP oxidation to p -quinone imine (PQI) at +290 mV followed by PQI reduction to PAP at −300 mV vs Ag/AgCl. Dual-electrode detection amplified the signal fourfold compared to single-electrode detection, and the recycling efficiency reached 87%. A calibration curve of PAP concentration vs anodic current was linear between 10 −4 and 10 −6 M. A signal from 1000 beads in a 20-μL drop was detectable and the immunoassay was complete within 10 min with a detection limit of 3.5 × 10 −15 mol mouse IgG.

This paper presents a new microfabricated plastic-based micro capillary electrophoresis (μ-CE) chip using a promising cyclic olefin copolymer (COC), which has wide range optical clarity down to the UV region. The μ-CE chips are fabricated using plastic injection molding techniques. The master mold for the μ-CE chip was fabricated on a nickel mold disk using thick photoresist processing and subsequent electroplating of nickel. The injection molded plastic chips were bonded using thermoplastic fusion bonding. In this study, a COC-based micro capillary electrophoresis (μ-CE) chip has been explored for the first time for separation of peptides and its experimental results are presented.

Hand-held, fully automated microfluidic systems have been developed and characterized to perform a magnetic bead-based biochemical immunoassay. The magnetic beads were coated with a polymer on which a dendritic antibody had been prepared to form the basis of a “sandwich” immunoassay for sensing biomolecules. With the press of a button, a complete immunoassay is performed to detect biohazards, and the result of the assay is displayed on a LCD. One system was made by using biocompatible silicone tubing for interconnections between the devices while another system was fabricated on a micromachined “glass-on-silicon” motherboard (Dharmatilleke et al.,. 2000). The first system consists of four collapsible reservoirs for reagent storage, made of very thin biocompatible Teflon™; an array of zero dead volume pinch valves; two magnetic curtain magnetic particle separators; a micro wire electrode or IDA for detection; a flow sensor, a valveless rotary pump to pull the reagents through the microfluidic system and associated electronics. On the second system, the mini pinch valves have been replaced by “collapsible” membrane type zero dead volume valves and pumping is by peristaltic operation of the valves. This work preceeds the next step, which is an all-MEMS system “on-a-chip”.

A multianalyte immunoassay concept based on the geometric separation of different analyte-specific antibodies has been demonstrated. The assay and amperometric detection are done in a cell with two working electrodes controlled at the same potential, and the amperometric signal at each electrode is monitored. The distance between any two adjacent electrodes in this prototype is 2.5 mm, and during the course of amperometric measurement, the product formed at one electrode does not reach the other working electrode within 20 min after the addition of enzyme substrate. Thus, the method relies on the spatial resolution between the different antibodies being such that measurements are taken before cross-interference due to diffusion can occur. Identical enzyme labels (alkaline phosphatase, ALP) and substrates (p-aminophenyl phosphate, PAPP) are used for all analytes. Alkaline phosphatase-conjugated rat anti-mouse IgG was immobilized by passive adsorption. Our studies showed that this concept is feasible and can be applied to the simultaneous measurement of multiple analytes.

A paramagnetic microbead-based enzyme immunoassay was demonstrated for detecting ovalbumin (OVA). The immunoassay sandwich was made by attaching a biotinylated antibody to the streptavidin coated beads as a mobile solid phase, capturing antigen, and then exposing the antigen to an antibody conjugated with β-galactosidase. β-Galactosidase converts p-aminophenyl galactopyranoside (PAPG) and fluorescein di-β-D-galactopyranoside (FDG) to p-aminophenol (PAP) and fluorescein, respectively. The current response of PAP generated by the enzymatic reaction of β-galactosidase was detected with hydrodynamic voltammetry in a droplet using a rotating disk electrode (RDE) system. The performance of this electrochemical assay was compared with fluorometric detection of fluorescein produced by the same assay system. The limits of detection for OVA determined by hydrodynamic amperometry and fluorometry were 2.1 nM (43 fmol) and 2.9 nM (58 fmol), respectively. Furthermore, the effects of the conditions commonly found in drinking water supply systems on the OVA assay were also evaluated.

Orosomucoid, a member of the lipocalin family, may function in the in vivo transport of lipophilic compounds such as basic and neutral drugs. We describe the identification of 7-chloro-1-methyl-1,5-benzodiazepine-2,4-dione (clobazam) bound to the serum orosomucoid from individuals actively taking this tranquillizer. This suggests not only that other endogenous factors limit access to the benzodiazepine binding site on human serum albumin, but also that the differential binding of benzodiazepines and their metabolites by orosomucoid should be considered in determining therapeutic doses, particularly in the acute phase response.

An amperometric method for alkaline phosphatase is described and compared to the most widely used spectrophotometric method. Catalytic hydrogenation of 4-nitrophenylphosphate (the substrate in the spectrophotometric method) gives 4-aminophenylphosphate (the substrate in the amperometric method). The latter substrate has the formula C6H6NO4PNa2.5H2O and a Mr of 323. The Michaelis constant for 4-aminophenylphosphate in 0.10 M, pH 9.0. Tris buffer is 56 microM, while it is 82 microM for 4-nitrophenyl phosphate. The amperometric method has a detection limit of 7 nM for the product of the enzyme reaction, which is almost 20 times better than the spectrophotometric method. Similarly, with a 15-min reaction at room temperature and in a reaction volume of 1.1 ml, 0.05 microgram/l alkaline phosphatase can be detected by electrochemistry, almost an order of magnitude better than by absorption spectrophotometry. Amperometric detection is ideally suited for small-volume and trace immunoassay.

This paper presents the development and characterization of an integrated microfluidic biochemical detection system for fast and low-volume immunoassays using magnetic beads, which are used as both immobilization surfaces and bio-molecule carriers. Microfluidic components have been developed and integrated to construct a microfluidic biochemical detection system. Magnetic bead-based immunoassay, as a typical example of biochemical detection and analysis, has been successfully performed on the integrated microfluidic biochemical analysis system that includes a surface-mounted biofilter and electrochemical sensor on a glass microfluidic motherboard. Total time required for an immunoassay was less than 20 min including sample incubation time, and sample volume wasted was less than 50 microl during five repeated assays. Fast and low-volume biochemical analysis has been successfully achieved with the developed biofilter and immunosensor, which is integrated to the microfluidic system. Such a magnetic bead-based biochemical detection system, described in this paper, can be applied to protein analysis systems.

The reduction potential of horse heart cytochrome c in various sodium halide solutions in H2O and D2O has been measured over the temperature range of 25 to 50 °C. In aqueous chloride solutions the temperature dependence was biphasic with the intersection point at 42 °C. This biphasic behavior is interpreted in terms of chloride-induced bulk destructuring of water at 42 °C. All samples in D2O and samples in H2O not containing chloride ion gave a linear temperature dependence. The decrease in reduction potential with increasing temperature for solutions containing F−, Cl−, Br− and I − correlates with the extent of anion binding to the oxidized form of cytochrome c.

Enzyme immunoassays based on chromatographic separation and amperometric detection of an enzyme generated product have been investigated. These assays combine the selectivity of the antigen/antibody reaction with the high sensitivity of thin layer amperometry. The feasibility of utilizing LCEC as a detection scheme was demonstrated using the Syva EMIT® kit for phenytoin. NADH production by glucose-6-phosphate dehydrogenase was monitored following a homogeneous procedure. Heterogeneous assays were developed for alkaline phosphatase labeled species which were based upon LCEC determination of phenol. Assays were designed for a common serum glycoprotein (orosomucoid) and a clinically important drug (digoxin). Detection limits approach the pg/mL level and as such may prove fruitful in the quantitation of numerous antigens of clinical interest.

Immunoassay is a very selective and sensitive approach to trace metabolite analysis (Thorell, 1978). As a consequence of binding geometries the interaction between antigen (Ag)† and antibody (Ab) molecules may be extremely specific under favorable conditions (Eisen, 1974). The utility of immunoassay in diagnostic medicine is well established with many routine clinical methods already in use for the determination of a wide variety of biologically important substances (Chalt and Ebersole, 1981).

The reaction between plasmin and alpha 2PI (alpha-2-plasmin inhibitor), which constitutes a major regulatory step in fibrinolysis, involves both interactions between the kringle structures of plasmin and a complementary site on the inhibitor, and also between the inhibitor reactive center and the enzyme active site. An attempt was made to distinguish calorimetrically the two functional domains of alpha 2PI. Only one transition was seen, both in the DSC and UV experiments, contrary to the expected two. This transition was unaffected by K4 of plasminogen but was abolished in the presence of anhydrotrypsin. This suggests the major domain structure in alpha 2PI is associated with the reactive center.

Enzyme immunoassays (EIAs) are methods which use the chemical amplification feature of an enzyme and the high specificity of an antibody to provide a highly sensitive and selective means for quantitating an analyte. The applications of EIAs are numerous and ever-expanding (Jarvis, 1979). The sensitivity of EIAs is as high as that of radioimmunoassays (Maggio, 1980) which have detection limits ranging from 1-500 fmol/mL assay fluid (Oellerich, 1980). EIAs can be classified as either heterogeneous or homogeneous.

Immunoassay is a powerful method exhibiting high selectivity and sensitivity for biologically important molecules (Chait and Ebersole, 1981). The utility of immunoassay in diagnostic medicine is well documented, and a number of routine clinical methods have been established for the determination of a wide variety of common antigens (Landon and Moffar, 1976). However, immunoassay has only recently gained prominence within the analytical community (Monroe, 1984).

A sandwich electrochemical enzyme immunoassay with flow injection analysis for the model antigen mouse IgG has been developed with alkaline phosphatase as the enzyme label. The enzyme substrate, 4-aminophenyl phosphate and its enzymatic reaction product, 4-aminophenol have been studied by cyclic and hydrodynamic voltammetry. The determination of 4-aminophenol by flow injection analysis with electrochemical detection (FIAEC) has a linear range of 5.0 × 10 −8 to 1.0 × 10 −5 M, a detection limit of 2.4 × 10 −8 M, and a sample throughput of 72 samples/h. The detection limit is set by a background capacitance response, which depends on the ionic strength difference between the sample and the mobile phase. The sandwich immunoassay has been characterized with respect to substrate concentration for the enzymatic reaction, detection limit, dynamic range and sources of error. Mouse IgG can be determined with a detection limit of 0.81 pg ml −1 by a 30-min substrate incubation time and a six orders of magnitude linear dynamic range.