William R. Heineman

An integrated microfluidic biochemical detection system for protein analysis with magnetic bead-based sampling capabilities

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.

p-aminophenyl phosphate: an improved substrate for electrochemical enzyme immnoassay

An alternative substrate is described for enzyme immunosaasay with electrochemical detection. Alkaline phosphatase (EC.3.1.3.1) activity is determined by using p-aminophenyl phosphate as the enzyme substrate. Enzyme-generated p-aminophenol is detected amperometrically at a glassy carbon electrody by liquid chromatography with electrochemical detection. The oxidation potential obtained for the detectionof p-aminophenol is lower than that for phenol, the previously used substrate product. The detection limit for p-aminophenol is 0.20pmol. A detection limit of 30 pg ml-1 for digoxin and a 5-min incubationtime for the enzyme reaction were obtained with the new system.

Environmental protection and economization of resources by electroorganic and electroenzymatic syntheses

The electrochemical methodology is an intrinsically environmentally friendly technique. It is especially excellently suited for preventive environmental protection because the practically mass-free electrons are used as reagents. Therefore, it allows the production of organic compounds without the formation of ecologically critical waste which has to be disposed. In addition, toxic waste formation can be prevented by continuous in situ or two-step electrochemical regeneration of heavy metal redox reagents. By using solid polymer electrolytes (ion-exchange membranes), even the use of a supporting electrolyte can be avoided. Thus, product formation can take place in pure methanol without any other chemical present. The consumption of resources can be economized by generating high-value products on both electrodes, anode and cathode (paired electrosynthesis). In certain cases, the same product may be formed on the anode and the cathode (200%-cell). Finally, in electroenzymatic syntheses, two environmentally friendly methods can be combined for the regeneration of the cofactors or the prosthetic groups of redox enzymes.

Revolutionizing biodegradable metals

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.

Nanotube electrodes and biosensors

This article reviews the state of the art in carbon nanotube electrode and biosensor research. Carbon nanotubes have unique mechanical, electrical, and geometrical properties that are ideal for developing different types of nanoscale electrodes and biosensors. Carbon nanotube synthesis and subsequent functionalization strategies to immobilize special biomolecules are discussed first. Then different types of carbon nanotube biosensors and electroanalytical methods are reviewed particularly considering their capabilities for low detection limits, point-of-care applications, and label-free use. Detection strategies for proteins and nucleic acids, as well as mammalian and bacterial cells are also outlined. We conclude with some speculations and predictions on future exciting and challenging directions for nanotube biosensor research and applications.

Development and Characterization of Microfluidic Devices and Systems for Magnetic Bead-Based Biochemical Detection

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.

Fast escape of hydrogen from gas cavities around corroding magnesium implants.

Magnesium materials are of increasing interest in the development of biodegradable implants as they exhibit properties that make them promising candidates. However, the formation of gas cavities after implantation of magnesium alloys has been widely reported in the literature. The composition of the gas and the concentration of its components in these cavities are not known as only a few studies using non-specific techniques were done about 60 years ago. Currently many researchers assume that these cavities contain primarily hydrogen because it is a product of magnesium corrosion in aqueous media. In order to clearly answer this question we implanted rare earth-containing magnesium alloy disks in mice and determined the concentration of hydrogen gas for up to 10 days using an amperometric hydrogen sensor and mass spectrometric measurements. We were able to directly monitor the hydrogen concentration over a period of 10 days and show that the gas cavities contained only a low concentration of hydrogen gas, even shortly after formation of the cavities. This means that hydrogen must be exchanged very quickly after implantation. To confirm these results hydrogen gas was directly injected subcutaneously. Most of the hydrogen gas was found to exchange within 1h after injection. Overall, our results disprove the common misbelief that these cavities mainly contain hydrogen and show how quickly this gas is exchanged with the surrounding tissue.

Comparison of methods for following alkaline phosphatase catalysis: Spectrophotometric versus amperometric detection

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.

Extending the detection limit of solid-phase electrochemical enzyme immunoassay to the attomole level

Electrochemical enzyme immunoassay methodology has been developed to take advantage of the selectivity of antibody reactions, the amplification feature of an enzyme-based assay, and the ease with which small amounts of the enzyme-generated product can be detected electrochemically. A heterogeneous sandwich enzyme immunoassay was used in this work as the model assay. In this type of assay, the antigen is sandwiched between the enzyme conjugate and a primary antibody that is adsorbed to the solid phase. Alkaline phosphatase is a suitable enzyme for electrochemical assays since it catalyzes the conversion of electroinactive phenyl phosphate to electroactive phenol. The product, phenol, is then quantitated by liquid chromatography with electrochemical detection in a thin-layer flow cell with a carbon paste electrode at 0.895 V vs Ag/AgCl. The current produced by the oxidation of phenol is directly proportional to the analyte (antigen) concentration. The problem associated with these types of solid-phase immunoassays is that the adsorption of the primary antibody is desired while the adsorption of other assay proteins is not. The detection limits are generally defined by the ability to control this nonspecific adsorption. The detection limit of a previous electrochemical assay for rabbit IgG was 100 pg/ml and was limited by a large background current observed in the absence of antigen. In the present study, each step of the assay was examined in order to determine the sources of this background current, and it was found that the major contribution was from the nonspecific adsorption of the enzyme conjugate. Using combinations of Tween 20 and bovine serum albumin as blocking agents, the level of nonspecific adsorption was reduced by 96%.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbohydrate-based label-free detection of Escherichia coli ORN 178 using electrochemical impedance spectroscopy.

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.