Novel Approaches in Biosensors and Rapid Diagnostic Assays

We have developed rapid, selective, and extremely sensitive assays for labeled and unlabeled oligonucleotides utilizing high-density fiber-optic microarrays. Microarrays are prepared by randomly distributing DNA-functionalized 3 µm-diameter microspheres in an array of wells etched in a 500 µm diameter optical imaging fiber. The fluorescence responses of the individual microsphere elements in the array are monitored by an imaging fluorescence microscope system. With labeled targets, hybridization was observed with a detection limit of 10 zmol DNA in 10 µL. With unlabeled targets, distinction between amplified genomic WT507 and AF508 Cystic Fibrosis samples was observed.

Acoustic wave devices of the transverse wave type offer tremendous potential for the study of biochemical macromolecules and cells imposed at the sensor liquid interface. Although this category of biosensor is conventionally thought of as a detector only, the device can also impart important structural and other information. The reason for this lies in the fact that such species are present on the device surface at precisely the location where solid-liquid coupling occurs. This means that the interfacial structure of biochemical entities can alter the propagation of acoustic energy in an exquisitely sensitive manner. With regard to protein adsorption, the competitive binding of blood proteins and antibodies to surfaces displaying different energy will be described. Such processes are important in the role played by polymers in the biocompatability of cardiovascular and breast implants. Our results indicate that a complete reevaluation of some accepted concepts in this area, including the well-known Vorman effect, is warranted. Also with respect to haematology the adhesion of blood platelets to various surface-bound receptors will be discussed. In this case the sensor responds to the viscoelastic properties of the cells rather than mass. Our most recent work on HIV transcriptional chemistry will be evaluated. Not only does the acoustic wave device sensitively detect peptide and small molecule binding, but the structural effects caused by different moieties can be discriminated. Finally, the use of enzymes in the real-time regeneration of singlestrand species of DNA attached to TSMs will be described.

A Surface Transverse Wave (STW) acoustic biosensor for monitoring chemical interactions in aqueous solution has been developed. The STW acoustic biosensor was utilized to monitor antigen-antibody and ligand-receptor interactions, and to follow sorption kinetics of organic chemicals dissolved in aqueous solution. The STW delay-line sensor has been designed to control the oscillation frequency of an rf oscillator at ~ 80 MHz. SiO₂ and poly[methylmethacrylate] (PMMA) served as wave guiding layers for the STW devices. The acoustic devices were placed in a thermostatic (25 °C) stainless steel flow-cell, and frequency has been followed with time. The applicability of the STW device in three model systems will be presented and discussed: The immunosensor model was based on the interaction between rabbit IgG and goat anti rabbit IgG peroxidase conjugate. The receptor-ligand interaction model was studied by use of a matrix bound procainamide and a soluble butyrilcholinesterase (BuChE). A special methacrylic-procainamide polymer (PPA) was designed and synthesized as an anchor for the BuChE molecule. Chemical sorption of organic solvents in aqueous phase was studied by poly[nbutylmethacrylate] (PBMA) coated STW sensors with SiO₂ as a wave guiding layer. Sorption kinetics were analyzed by a simulation method to derive the chemical diffusion and partition coefficients.

Amplified electronic transduction of DNA sensing is accomplished by the application of a biocatalytic probe that precipitates an insoluble product on the transducer as a result of the DNA sensing. Alternative amplification of DNA sensing is achieved by the use of functionalized liposomes that bind to the electrode upon the sensing process. Single base mutations in DNA are detected by the polymerase-induced coupling of a biotinylated base complementary to the mismatch site, followed by the biocatalyzed precipitation of an insoluble product on the transducer. DNA biosensors of unprecedented sensitivity and specificity are designed. Faradaic impedance spectroscopy and microgravimetric quartz-crystal-microbalance measurements are used for the electronic transduction of the DNA sensing.

A single base pair mismatch in an 18 base oligonucleotide was detected with a 7 µm diameter carbon microelectrode. The hybridization was followed directly and in real time by steady state amperometry. The microelectrode was coated with a hybridization-sensing layer in a two-step electrophoretic process, which yielded microelectrodes with reproducible dimensions. In the first step, a thin film of an electron-conducting redox polymer was deposited electrophoretically at constant potential in a low ionic strength solution. In the second step a carbodiimide activated single stranded probe was reactively electrophoretically deposited and covalently attached to the redox polymer film. The labeling enzyme, thermostable soybean peroxidase (SBP), was covalently bound to the 5’-end of the target single stranded oligonucleotide. When the redox polymer and the enzyme were brought to close proximity by hybridization of the target and probe oligonucleotides, the film on the electrode switched from being a non-catalyst to a catalyst for H₂O₂ electroreduction at -0.06 V vs. Ag/AgC1. The current observed corresponded to that generated by approximately 40,000 surface bound and electrically connected SBP molecules.

This work describes the development and application of rapid, disposable and sensitive bioelectrochemical sensors. The sensors are based on enzyme amplification amperometric detection, with screen-printed electrodes, a microflow injection system and an enzymatic membrane placed in a microflow electrochemical cell. The basic configuration of the sensor can be adapted to and applied in various analytical determinations. Examples of sensors to detect organophosphorous pesticides, formaldehyde and lactose in raw milk are discussed.

Applications of sol-gel derived carbon-ceramic electrodes (CCEs) as biosensors are reviewed. CCEs are comprised of a dispersion of carbon grains in sol-gel silicates, organically modified silicates, or other metal oxide binding networks. Inorganic, organic or biochemical moieties can be incorporated by sol-gel doping, impregnated on pre-prepared CCEs, or introduced as organofunctional groups of the sol-gel monomer. Enzyme, tissue cell and immunosensor based CCE biosensors are described.

The concept of using a simple reflection hologram as both an analyte-sensitive polymer matrix and an optical interrogation and reporting transducer to generate inexpensive, mass-producible biochemical sensors is descibed. The system is exemplified with holographic sensors for water and enzyme activity and the value of rationally designed holographic matrices promoted.

A highly-sensitive surface fluorescence technique was combined with a powerful method for the oriented reversible immobilization of membrane proteins on oxide surfaces. Affinity and kinetic parameters of the interaction of the receptor and a fluorescent ligand were evaluated. The approach is particularly suited for the study of rapid ligand-receptor interactions where optimized cell volume, flow and injection conditions are required.

Although tobacco smoking is widespread in the population and some of its toxic effects are widely recognized the mechanisms underlying nicotine addiction and its toxicity still remain poorly understood. An important step toward this understanding was made with functional studies of nicotinic receptors reconstituted in host systems. However, it becomes increasingly important to understand their role in more physiological conditions. Namely, it is mandatory to be able to examine their contribution in a natural neuronal network. One of the best possible approaches would be to dispose of multiple electrode recordings that would allow investigation of acute brain slices. While planar multielectrode arrays constitute one possibility for multiple recordings of neurones in culture their signal noise ratio is, however, not sufficient for acute slices. To overcome this problem we are currently developing a “fakir bed” electrode array in which the electrode tips are brought in closer contact with intact neurones within the slice. In addition to being expressed by neurones, nicotinic acetylcholine receptors are also expressed in other cells throughout the body. For instance it has been shown that white blood cells express significant amount of these proteins and that nicotine can modulate the motility abilities of leukocytes. In a series of experiments we have been able to study these cellular processes in more detail and have established a new biochip technology with the aim of automatic evaluation of cell motility.

DNA-microarrays have become a synonym for the type of analyses that aim at the understanding of cellular functioning in a comprehensive manner. Although still in a relatively early phase, the methodology has already proven its worth. Nevertheless, improvements on various aspects are necessary for making DNA-chips a routine tool in research and diagnostics. In this manuscript, some chemical developments toward this end are being reviewed.

This chapter presents some reflections on biorecognition and describes its use in biosensors and affinity techniques, such as affinity chromatography, immunoaffinity chromatography, (photo)affinity labeling, affinity cross-linking, and affinity therapy.

Molecular imprinting is a technique that permits the synthesis of polymers containing specific recognition sites for a target molecule through the use of templates. This paper describes recent developments in the design and optimization of molecularly imprinted polymers, and their application as the recognition element in chemical or biomimetic sensors and assay systems. The work presented is mainly emanating from our own research group.

Optical and mass-sensitive transducers coated with synthetic antibodies allow analyte detection in liquid phase. Flow cells for a quartz crystal microbalance (QCM) with screen printed multi-electrode structures offer excellent temperature/viscosity compensations and automation potentials. Imprinting polyurethanes preferably with binary mixtures of polyaromatic hydrocarbons (PAHs) at elevated temperatures makes possible a tuning of the sensor selectivity. Thus, detection limits down to some ng/L are accessible and crosssensitivities to humic acids are eliminated. Furthermore, copolymers including acrylic or methacrylic acid form recognition sites for xanthine derivatives after caffeine imprinting. The complex process of automotive oil degradation is monitored with fresh and used-oil imprinted polyurethanes and sol-gel phases. Compensating for viscosity changes during oil ageing guarantees the characterization of the pure chemical changes.

A new biotinylated-bipyrrole derivative has been synthesized and its coelectropolymerisation with pyrrole developed for the immobilization of ligands onto electrode surfaces. The biotin-polymeric film was produced on a gold electrode and the FITC-avidin binding to the film surface was demonstrated by fluorescence detection using an epifluorescent microscope.

DNA-microarrays have become a synonym for the type of analyses that aim at the understanding of cellular functioning in a comprehensive manner. Although still in a relatively early phase, the methodology has already proven its worth. Nevertheless, improvements on various aspects are necessary for making DNA-chips a routine tool in research and diagnostics. In this manuscript, some chemical developments toward this end are being reviewed.

Uniform latex particles or microspheres were first used in medical diagnostic applications as “latex” agglutination tests (LAT). Sensitive particle-enhanced turbidimetric assays are still in common use and are read with clinical chemistry analyzers via spectrophotometric or nephelometric methods. Dyed agglutinated particles caught on filters form the basis of another class of tests. Particle capture ELISA tests and assays are in common use (e.g., Abbott’s IMx and AxSym). The popular “strip tests” for pregnancy, ovulation, drugs of abuse in urine, etc. use dyed microspheres, and quantitative strip assays are beginning to appear. Solid phase assays and tests use particles for positive or negative capture of a wide variety of analytes. Solid-liquid separations can be made by centrifugal density separation, or filtration, or via magnetic separation of superparamagnetic particles. Proximity assays, like scintillation proximity assay (SPA), luminescent oxygen channeling immunoassay (LOCI), and fluorescence resonance energy transfer (FRET), all use microspheres. Single microsphere assays are now possible in flow cytometers and on the newer flow-based analysers. Dyed microspheres can be much more sensitive as stains or markers since a single 100 nm microsphere can carry 1000 dye molecules. Molecular biology applications include the Human Genome Project where superparamagnetic and silica microspheres are used to separate DNA from cell debris. Microspheres have been used in immunosensors based on piezoelectrics and evanescent-wave optical fiber-based immunosensors. One new assay system currently being developed uses single beads caught in wells etched in the ends of optical fibers.The hottest new immunological use for microspheres is in homogeneous multiplexed pharmaceutical high-throughput screening assays.

A novel homogeneous luminescence detection method, based on singlet oxygen channeling, LOCI ™, was recently described (1,2). The utility of the new method for detection and quantification of small molecules, high molecular weight molecules such as nucleic acids and proteins, complex structures such as specific cell surface and viral components, as well as detection of nucleic acid sequence alteration, will be presented.

Photodynamic Therapy (PDT) is an anti-cancer treatment modality in which sensitizer drug, light and oxygen are used to photochemically cause cell death. The cytotoxicity of PDT is based on the toxic effects of singlet oxygen and other free radicals generated by the photosensitizer in target cells upon illumination. Successful application of anticancer therapy and especially PDT depends on oxygen content within the tumor. Application of an optical oxygen microsensor allowed continuous and direct in situ measurements of temporal variations in the oxygen partial pressure (pO₂) during PDT. The oxygen microsensor, developed by us in collaboration with OST, Israel, is an on-line system based on fluorescence measurements that provides continuous monitoring of blood and tissue oxygen levels. This study examined the spatial and temporal variations in the concentration of oxygen within solid tumors, during PDT. We found that illumination of Bacteriochlorophyll-Serine (a sensitizer drug) treated tumors leads to a rapid decline in tissue oxygen. Analysis of the process permitted a detailed description of the dynamics of oxygen depletion and reoxygenation. Moreover, it was shown that tumor oxygen tension becomes irreversibly low as a result of the effect of PDT. These results were interpreted in terms of vasculature damage. Magnetic resonance imaging studies confirmed the above findings and clearly showed that PDT induces vascular damage. Integration of the sensor in PDT experiments enables the in vivo analysis of the basic mechanisms underlying photoinduced cytotoxicity as well as the development of treatment protocols that optimize the synchronization of light pulses with tissue reoxygenation.

Glucose can be extracted through intact skin upon application of a low-level electrical current by electroosmotic flow (a process called “reverse iontophoresis”). Recently we have combined iontophoretic extraction with an in situ glucose sensor in a device called the Gluco Watch^® biographer. Clinical results with this device show close tracking of blood glucose over a range of 40 to 400 mg/dL (2.2 to 22.2 mmol/L) for up to 12 hours using a single blood glucose value as calibration. The biographer readings lag behind blood glucose values by an average of 18 minutes. An analysis of data from 92 diabetic subjects in a controlled clinical setting shows a linear relationship (r=0.88) between GlucoWatch biographer readings and blood glucose. The mean absolute difference between the two measurements was 15.6% and more than 96% of the data fell in the (A+B) regions of the Clarke error grid. Similar results have been obtained from subjects using the GlucoWatch biographer in an uncontrolled home environment. The automatic, frequent, and non-invasive measurements obtained with the GlucoWatch biographer provide substantially more information about glucose levels than the current finger-stick methods. This information can be used for improved decisions about all aspects of diabetes management.

Rapid Methods and Automation in Microbiology is a dynamic field dealing with all aspects in the isolation, enumeration,characterization and identification of microbes from water, food, clinical specimens, industrial and environmental samples. This communication highlights the recent developments in sample preparations, efficient methods for viable cell counts, instrumental methods for the estimation of microbial populations and biomass, miniaturized microbiological techniques, modern immunological procedures, and advanced genetic methods. The aim is to rapidly acquire microbiological information from target samples so that effective measures can be taking for consumer protection.

The threat of attack on military and civilian targets with chemical and biological weapons is a growing national concern. The Defense Advanced Research Projects Agency (DARPA) is developing technologies for detecting biological materials in the natural environment. While several technologies show promise as broadband detectors, there is no “silver bullet” that detects all chemical and biological materials at the requisite levels of sensitivity and specificity. DARPA is developing a systems approach whereby several different advanced detection schemes (based on different physical phenomena) are being integrated into a biological detection suite.