Chemical sensors based on controlled-release polymer systems.

A novel chemical sensor has been developed in which the polymer ethylene-vinyl acetate is used as a controlled-release system to deliver reagents to the sensing region of an optical fiber for a homogeneous competitive immunoassay based on fluorescence energy transfer. A competition reaction is used to enable continuous measurements of the solution antigen concentration. More generally, the technique allows irreversible indicating chemistries to be used in the construction of chemical sensors that can measure continuously for long periods. Although the sensor configuration has not been optimized in all respects, data are presented for a model system in which a fluorescein-labeled antibody and Texas Red-labeled immunoglobulin G (IgG) are used.

Photodeposition of micrometer-scale polymer patterns on optical imaging fibers.

Microstructures were fabricated on optical imaging fibers with a photopolymerization technique. Monodisperse polymeric microarrays were produced containing spots of 2.5 micrometers in diameter spaced 4.5 micrometers apart. Polymer microarrays were also deposited on other substrates by using imaging fibers for light delivery. The technique allows micrometer-scale photopatterning with masks larger than the desired dimensions.

Screening unlabeled DNA targets with randomly ordered fiber-optic gene arrays.

We have developed a randomly ordered fiber-optic gene array for rapid, parallel detection of unlabeled DNA targets with surface immobilized molecular beacons (MB) that undergo a conformational change accompanied by a fluorescence change in the presence of a complementary DNA target. Microarrays are prepared by randomly distributing MB-functionalized 3-microm diameter microspheres in an array of wells etched in a 500-microm diameter optical imaging fiber. Using several MBs, each designed to recognize a different target, we demonstrate the selective detection of genomic cystic fibrosis related targets. Positional registration and fluorescence response monitoring of the microspheres was performed using an optical encoding scheme and an imaging fluorescence microscope system.

A fiber-optic DNA biosensor microarray for the analysis of gene expression.

A fiber-optic biosensor array is described for the simultaneous analysis of multiple DNA sequences. A bundle of optical fibers was assembled with each fiber carrying a different oligonucleotide probe immobilized on its distal end. Hybridization of fluorescently labeled complementary oligonucleotides to the array was monitored by observing the increase in fluorescence that accompanied binding. The approach enables fast (< 10 min) and sensitive (10 nM) detection to multiple DNA sequences simultaneously, with the potential for quantitative hybridization analysis.

Enzyme Kinetics in Femtoliter Arrays.

Over the last decade, femtoliter arrays have been used as a simple and robust way to encapsulate and monitor the kinetics of single enzyme molecules. Encapsulating individual enzyme molecules within a femtoliter-sized reaction chamber does not require immobilization of the enzyme molecules or fluorescent tagging of the enzyme molecules, which offers the unique advantage of observing unmodified single enzyme molecules free in solution. Several fascinating details about enzyme kinetics have been revealed using these femtoliter arrays, which were unattainable from traditional ensemble experiments. Here, we discuss various considerations to take into account when developing single-molecule enzyme assays in femtoliter arrays and the advantages and disadvantages of various protocols.

Current trends in 'artificial-nose' technology.

Basic principles derived from biological olfaction, such as combining semiselective sensor arrays with pattern recognition, have been used to develop instrumentation capable of broad-band chemical detection and quantification. Commercially available instruments are useful in areas including quality control in the food, beverage and fragrance industries, environmental monitoring, chemical-purity and -mixture analysis, and medical diagnostics. Ongoing research is aimed at the development of more-advanced instruments that are smaller, cheaper, faster and more stable and reliable. These second-generation instruments are likely to find an increasing number of applications, including the on-line monitoring of fermentation and other bioprocesses.

Combined imaging and chemical sensing of L-glutamate release from the foregut plexus of the lepidopteran, Manduca sexta.

A new combined imaging and chemical detection sensor for the measurement of localized L-glutamate release at the insect neuromuscular junction (NMJ) is presented. The sensor is comprised of an L-glutamate-sensitive fluorescent gel, spin-coated onto the tip of an optical imaging fiber. The gel is composed of L-glutamate oxidase (GLOD); a pH-sensitive fluorescent dye, SNAFL; and poly(acrylamide-co-N-acryloxysuccinimide) (PAN). NH(3) is liberated from the interaction of L-glutamate with GLOD, which reversibly reduces the emitted fluorescence signal from SNAFL. This sensor has a spatial resolution of 3-4 micro m, and an L-glutamate detection limit of between 10 and 100 micro M. L-glutamate release and re-uptake from the foregut plexus of Manduca sexta was detected by the sensor in the presence of the L-glutamate re-uptake blocker dihydrokainate, and the post-synaptic L-glutamate receptor antagonist CNQX.

Multianalyte biosensors on optical imaging bundles.

We present an optical biosensor design that expands the utility of enzyme biosensors. These biosensors are fabricated by site-selective photodeposition of analyte-sensitive polymer matrices on optical imaging fibres. These dual-analyte arrays allow for the simultaneous, independent measurement of the analyte of interest and the transducing analyte. The first integrated optical-biosensors using this design have been prepared that allow both the dependent and independent analytes to be measured simultaneously, for example penicillin and pH (Healey & Walt, 1995) or glucose and O2 (Li & Walt, 1995). Independent measurement of the transducing analyte allows penicillin or glucose to be quantitated in the presence of a concurrent pH or O2 change, respectively. Penicillin can be measured in the range 0.25-10.0 mM in the pH range 6.2-7.5. Glucose can be measured in the range 0.6-20.0 mM in the O2 range 20-100%. The utility of the sensor design was demonstrated by using the penicillin-dual-analyte biosensor to quantitate penicillin produced during a Penicillium chrysogenum fermentation.

A fiber-optic lactate sensor based on bacterial cytoplasmic membranes.

A new type of fiber-optic biosensor based on bacterial cytoplasmic membranes (CPM) as the biological recognition element and an oxygen sensitive dye layer as the transducer is described for the detection of lactate. CPMs from bacteria with an induced lactate oxidase system are adsorbed onto a cellulose disk. The disk is fixed mechanically over an oxygen sensitive siloxane layer on the distal end of an optical fiber. This system detects lactate with no interference from glucose, fructose or glutamic acid.

Optical sensor arrays for odor recognition.

Optical sensor arrays containing fluorescent solvatochromatic dyes immobilized in a plurality of polymers generate information-rich responses upon exposure to organic vapors. The response profiles are used to train a variety of computational networks such that subsequent exposure of the array to the vapors enables them to be classified and/or quantified. A number of strategies can be taken to enhance sensitivity and to increase sensor diversity.

Fluorescent excitation transfer immunoassay for the determination of spinosyn A in water.

A fluorescent excitation transfer immunoassay for spinosyn A, a fermentation derived insect control agent, has been developed and applied to the analysis of tap water and wastewater effluent from manufacturing plants. Fluorescein (F) and tetramethylrhodamine (TMR) were chosen as donor and quencher, respectively, for the excitation transfer. Fluorescence quenching was observed from the binding of F-labeled antigen to TMR-labeled antibody. By employing nonlabeled antigen in a competitive immunoassay format, we reversed fluorescence quenching. The assay provides a limit of detection of 0. 01 ppb and a working range of 0.05-1 ppb and allows for the rapid determination of spinosyn A in water with recovery values ranging from 96% to 120%. With the exploitation of the small size of optical fibers, fluorescence from an assay volume of 24 microL could be measured without special vessels.

A fiber-optic sensor for CO(2) measurement.

A fiber-optic sensor has been prepared which responds to carbon dioxide at physiologically significant concentrations. It is based on pH modulation by dissolved carbon dioxide in a sensing layer of fluorescent dye. By use of a previously developed methodology by which the sensing chemistry is bonded directly to the glass fiber tip, the miniature size of the sensor is preserved. This method involves consecutive applications of solution polymers to the fiber tip rather than mechanical attachment of sensor reagents. Preparations of polymer-immobilized dyes and polymer membranes are described.

Fluorescent optical sensors.

Optical sensors are prepared by immobilizing an indicating layer on the distal end of a fiber optic cable. Dyes, enzymes, and antibodies can all be incorporated into the layer using a variety of immobilization techniques. Much of the present work is devoted to developing novel indicating schemes by combining appropriate recognition schemes into polymeric matrices.

Fiber-optic sensor for continuous monitoring of fermentation pH.

We have developed a fiber-optic chemical sensor for on-line monitoring of fermentation pH. The sensor is based on a covalently bound fluorescent dye immobilized within a water-permeable polymer layer on an optical fiber. Measurements were performed on a portable fluorimeter and employed a ratiometric approach to account for system instabilities. We show that the use of this fiber-optic sensor provides fast, accurate and reliable measurements during E. coli fermentation in a complex medium.

Whole-saliva proteolysis and its impact on salivary diagnostics.

There is growing interest in the use of human whole saliva for diagnostics and disease monitoring as an alternative to blood samples. In contrast to blood, whole saliva is a non-sterile body fluid. Proper hand-ling and storage are required to preserve the integrity of potential biomarkers. We investigated salivary autoproteolytic degradation using a variety of approaches. We determined inhibition of protease activities by monitoring the endogenous proteome. In addition, the stability of highly protease-susceptible proteins-histatin 5, statherin, and PRP1-was assessed. Experimental variables included (a) protease inhibitors, (b) salivary pH, (c) incubation temperatures, and (d) sample heating. A cocktail containing AEBSF, aprotinin, pancreatic trypsin inhibitor, leupeptin, antipain, and EDTA could not prevent histatin 5, statherin, or PRP1 degradation in whole saliva. Among the other treatments evaluated, short-term storage of freshly collected samples on ice was effective without interfering with the chemistry of the proteome. In conclusion, whole saliva contains a unique mixture of enzymes as evidenced from their resilience to protease inhibition. Analytical evidence on protein stability is needed to ensure the validity of salivary biomarker study outcomes. Analysis of the data presented will provide help and guidance for the use of saliva samples for diagnostic purposes.

Improved fiber-optic chemical sensor for penicillin.

An optical penicillin biosensor is described, based on the enzyme penicillinase. The sensor is fabricated by selective photodeposition of analyte-sensitive polymer matrices on optical imaging fibers. The penicillin-sensitive matrices are fabricated by immobilizing the enzyme as micrometer-sized particles in a polymer hydrogel with a covalently bound pH indicator. An array of penicillin-sensitive and pH-sensitive matrices are fabricated on the same fiber. This array allows for the simultaneous, independent measurement of pH and penicillin. Independent measurement of the two analytes allows penicillin to be quantitated in the presence of a concurrent pH change. An analysis was conducted of enzyme kinetic parameters in order to model the penicillin response of the sensor at all pH values. This analysis accounts for the varying activity of the immobilized penicillinase at different pH values. The sensor detects penicillin in the range 0.25-10.0 mM in the pH range 6.2-7.5. The sensor was used to quantify penicillin concentration produced during a Penicillium chrysogenum fermentation.

Fabrication of patterned sensor arrays with aryl azides on a polymer-coated imaging optical fiber bundle.

Arrays of sensing regions are photodeposited on the distal tip of a single imaging optical fiber. First, the distal surface of the fiber is spin-coated with a thin film of poly(hydroxyethyl methacrylate). The fluorophor is then derivatized with a photoreactive group and subsequently immobilized in a finite area of the film by discrete illumination. Dye incorporation occurs only in the illuminated areas, creating distinct regions of analyte-sensitive fluorescent dye at the fiber's distal end. This paper describes both the chemistry and the manipulations required to make an optical microarray and demonstrates the technique with pH sensors. The fabrication of a four-sensor array is described along with performance data.