Jerome S. Schultz

Affinity Sensor: A New Technique for Developing Implantable Sensors for Glucose and Other Metabolites

We describe affinity sensors for monitoring various metabolites in blood plasma by optical means. The principle of detection is similar to that used in radioimmunoassays and is based on the competitive binding of a particular metabolite and a fluorescein-labeled analogue with receptor sites specific for the metabolite and the labeled ligand. This concept has been directed toward the development of an affinity sensor for glucose. Concanavalin A, a protein with specific binding character for glucose, was immobilized on the inside surface of a hollow dialysis fiber. Fluorescein-labeled dextran was selected as the competitive labeled ligand. The molecular weight cutoff of the dialysis fiber is low enough to completely retain the 70,000 MW dextran within the fiber lumen while glucose can freely pass through the dialysis membrane. The sensor is completed by inserting a single optical fiber in the lumen of the dialysis fiber, thus allowing measurement of the unbound FITC-dextran. Preliminary tests of the sensor indicated the feasibility of the approach. Sensitivity to glucose in the physiologic range was obtained, but further work will be required to optimize the sensitivity and response time of the sensor.

Handbook of Chemical and Biological Sensors

PREFACE FUNDAMENTALS OF SENSOR TECHNOLOGY: INTRODUCTION TO CHEMICAL AND BIOLOGICAL SENSORS by J.S. Schultz and R. F. Taylor PHYSICAL SENSORS by R.A. Peura and S. Kun Piezoelectric sensors Resistive sensors Inductive sensors Capacitive sensors Bridge circuits Displacement measurements Blood pressure measurements INTEGRATED CIRCUIT MANUFACTURING TECHNIQUES APPLIED TO MICROFABRICATION by M. Madou and H.L. Kim Photolithography Subtractive techniques Additive techniques Comparison of micromachining tools Acknowledgment PHOTOMETRIC TRANSDUCTION by D.G. Buerk Phototransduction based on interactions between light and matter Applications for photometric transducers ELECTROCHEMICAL TRANSDUCTION by J. Wang Amperometric transduction Potentiometric transduction Conductometric transduction MODIFICATION OF SENSOR SURFACES by P. Barlett Covalent modification of surfaces Self-assembled monolayers and adsorption Polymer-coated surfaces Electrochemically generated films Other surface modifications BIOLOGICAL AND CHEMICAL COMPONENTS FOR SENSORS by J.S. Schultz Sources of biological recognition elements Design considerations for use of recognition elements in biosensors IMMOBILIZATION METHODS by R.F. Taylor Immobilization technology Immobilization of cells or tissues BILAYER LIPID MEMBRANES AND OTHER LIPID-BASED METHODS by D.P. Nikolelis, U.J. Krull, A.L. Ottova, and H.T. Tien Experimental bilayer lipid membranes Electrostatic properties of lipid membranes Electrochemical sensors based on bilayer lipid membranes BIOMOLECULAR ELECTRONICS by F.T. Hong Advantages of using molecular and biomolecular materials Electrical behavior of molecular optoelectronic devices: the role of chemistry in signal generation The physiological role of the ac photoelectric signal: the reverse engineering visual sensory transduction process Bacteriorhodopsin as an advanced bioelectronic material: a biunctional sensor Bioelectronic interfaces Immobilization of protein: the importance of membrane fluidity The concept of intelligent materials SENSOR AND SENSOR ARRAY CALIBRATION by W.P. Carey and B.R. Kowalski Zero-order sensor calibration (individual sensors) First-order sensors (sensor arrays) Second-order calibration MICROFLUIDICS by J.N. Zemel and R. Furlan Fabrication of small structures Sensors for use in microchannels Flow actuation and control Fluid flow phenomena PRACTICAL EXAMPLES OF POLYMER-BASED CHEMICAL SENSORS by M.J. Tierney Roles of polymers in chemical, gas, and biosensors Property/function-based selection of polymers for sensors Polymer membrane deposition techniques Examples: polymers in fast-response gas sensors SOLID-STATE, RESISTIVE GAS SENSORS by B. Hoffheins Materials Enhancing selectivity Fabrication Specific sensor examples OPTICAL SENSORS FOR BIOMEDICAL APPLICATIONS by G.G. Vurek Why blood gas monitoring? Oximetry Intra-arterial blood gas sensors Sensor attributes affecting performance Accuracy compared to what? Tools for sensor development Examples of sensor fabrication techniques In vivo issues ELECTROCHEMICAL SENSORS: MICROFABRICATION TECHNIQUES by C-C Liu General design approaches for microfabricated electrochemical sensors Metallization processes in the microfabrication of electrochemical sensors Packaging Practical applications Examples ELECTROCHEMICAL SENSORS: ENZYME ELECTRODES AND FIELD EFFECT TRANSISTORS by D. Pfieffer, F. Schubert, U. Wollenberger, and F.W. Scheller Overview of design and function Description of development steps Transfer to manufacturing and production Practical use and performance ELECTROCHEMICAL SENSORS: CAPACITANCE by T.M. Fare, J.C. Silvia, J.L. Schwartz, M.D. Cabelli, C.D.T. Dahlin, S.M. Dallas, C.L. Kichula, V. Narayanswamy, P.H. Thompson, and L.J. Van Houten Contributions to conductance and capacitance in device response Mechanisms of sensor response: kinetics, equilibrium, and mass transport Practical example: fabrication and testing of SmartSense immunosensors PIEZOELECTRIC AND SURFACE ACOUSTIC WAVE SENSORS by A.A. Suleiman and G.G. Guilbault Fundamentals Commercial devices Emerging technology THERMISTOR-BASED BIOSENSORS by B. Danielsson and B. Mattiasson Instrumentation Applications ON-LINE AND FLOW INJECTION ANALYSIS: PHYSICAL AND CHEMICAL SENSORS by G.E. Pacey Definitions and descriptions of on-line and flow injection Selectivity enhancements, matrix modification, and conversion Sensor cell design in FIA Measurements FLOW INJECTION ANALYSIS IN COMBINATION WITH BIOSENSORS by B. Mattiasson and B. Danielsson Flow injection analysis CHEMICAL AND BIOLOGICAL SENSORS: MARKETS AND COMMERCIALIZATION by R.F. Taylor Development and commercialization Current and future applications Current and future markets Development and commercialization of a chemical sensor or biosensor

HINDRANCE OF SOLUTE DIFFUSION WITHIN MEMBRANES AS MEASURED WITH MICROPOROUS MEMBRANES OF KNOWN PORE GEOMETRY

Abstract 1. 1. Mica sheets were made into membranes by a process of bombardment with fission fragments from a U235 source and subsequent etching with hydrofluoric acid. Pores formed by this process were essentially straight through the membrane, extremely uniform in size and elliptical in cross-section. On eight of these membranes, with pore radii ranging from 45 to 300 A, air flow, water flow, and diffusion rates for a graded series of 7 solutes were measured. From measurements of the diffusion rate of mostly non-electrolytes, with radii between 2.5 and 22.5 A, the true hindrance effect on diffusion within pores was determined. 2. 2. Restriction of diffusion for even relatively small solutes is a very significant effect and can be adequately described by the Renkin equation D m / D 0 = (1−R s /R p 2 ×(1−2.104 R s /R p + 2.09 (R s /R p −0.95 (R s /R p ) 5 ) for membranes which have well-defined, straight through pores, where D m / D 0 is the ratio of solute diffusivity in the membrane to that in free solution, and Rs/Rp is the ratio of solute radius to pore radius. An approximation to the Renkin equation, D m / D 0 = (1−R s /R p ) 4 , which is much simpler to use, correlates as well with the data in the range 0 3. 3. Water flow under small pressure drops is well described by the assumption of Poiseuille flow for this range of pore diameters, and therefore no effects due to “anomalous” water were apparent. 4. 4. In most membrane operations there is a considerable resistance to diffusion due to the presence of a liquid film boundary layer along the surface of the membrane. This boundary layer resistance was not inversely proportional to the solute diffusivity as has often been assumed in the “unstirred layer” theory, but instead was found under these experimental conditions to be proportional to the −0.6 power of the solute diffusivity. 5. 5. Boundary layer diffusion resistances were obtained by two independent methods: an electrochemical polarographic method and a membrane substitution method using membranes of known permeability to calibrate the diffusion cell. 6. 6. Heteroporous membranes do not differ from isoporous membranes very much in regard to the relative hindrance of solute molecules, as long as the ratio of solute radius to mean pore radius is less than 0.2. For larger solute molecules, heteroporous membranes become increasingly less effective in hindering diffusion rates through the membrane.

Design, manufacture and characterization of an optical fiber glucose affinity sensor based on an homogeneous fluorescence energy transfer assay system

Abstract Optical fiber biosensors based on fluorescence assays have several distinct advantages when measuring biological analytes such as metabolites, cofactors, toxins, etc. Not only are optical signals immune to electronic interferences, but the polychromatic nature of most fluorochemical assays provides more potentially useful data about the system being studied. One of the most common difficulties normally encountered with optical biosensors is the inability to routinely recalibrate the optical and electronic components of the system throughout the life of the sensor. With this in mind, an optical biosensor system for glucose has been constructed along with the peripheral electronic instrumentation. The biochemical assay is based on an homogeneous singlet/singlet energy transfer affinity assay. The sensor probe indirectly measures glucose concentrations from the level of fluorescence quenching caused by the homogeneous competition assay between TRITC labeled concanavalin A (receptor) and FITC labeled Dextran (ligand). The FITC signal is used as an indicator for glucose concentrations and the TRITC signal is used for internal calibration. Chemical derivatization procedures using succinic anhydride were developed to prevent aggregation of the receptor protein in solution, and the TRITC/ConA ratios were optimized to achieve the best sensor performance. Using this sensor system, the FITC-Dextran detection limit was 0.05 μg/ml and glucose concentrations up to 1600 mg/dl could be detected with a time response of approximately 10 min.

Fiber-optic biosensors based on fluorescence energy transfer

A new optical homogeneous biochemical method for the assay of glucose has been developed, based on fluorescence energy transfer between a glucose analog, dextran labeled with fluorescein isothiocyanate (FITC-dextran), and a glucose-receptor protein, Rhodamine-labeled Concanavalin A (Rh-ConA). When FITC-dextran binds to Rh-ConA in solution, and is light-activated, the FITC label transfers its absorbed energy to the Rhodamine label, which then emits light according to its own characteristic fluorescence spectrum. When glucose is added to this solution, the FITC fluorescence intensity increases as FITC-dextran is released from the Rh-ConA and is replaced by glucose. Thus it is possible to determine glucose concentrations directly from the level of FITC fluorescence.

Competitive-binding assay method based on fluorescence quenching of ligands held in close proximity by a multivalent receptor

Abstract A variant of a fluorescence quenching affinity assay is described that is based on intermolecular complexation due to specific interaction between an unmodified multivalent lectin and fluorochrome-labeled dextrans bearing specific sugar ligands (analyte-analog). The measuring principle relies on the fact that one portion of the dextran is coupled with an emitter dye fluorescein isothiocyanate (FITC), and the other one with an acceptor dye (isothiocyanate-derivatives of rhodamine). In absence of a specific sugar, the bridging of rhodamine and fluorescein-labeled dextrans by the lectin results in the formation of a sandwich-like fluorescein-dextran/lectin/rhodamine-dextran complex in which the two forms of dextran are very close together (~5 nm) so that fluorescence resonance energy transfer (FRET) occurs between fluorescein and rhodamine. Hence the fluorescence is quenched. The displacement of dextrans by a specific sugar results in the dissociation of the complex and in an inverse increase in fluorescence which is proportional to the sugar concentration. The paper describes experiments proofing the conceptual idea of this fluorescence assay on two examples: a glucose and galactose-specific assay system. The glucosespecific assay consisted of Concanavalin A (Con A) and fluorescein and rhodamine-labeled dextran (Mr 2000 kDa) grafted with mannose. The galactose-specific assay was composed of Ricinus communis agglutinin (RCAI) and fluorescein and rhodamine-labeled dextran (Mr 2000 kDa) grafted with lactose. The reversibility and response time of both assays inside a single dialysis hollow fiber, which was fixed within a flow through cell of a fluorometer, were studied during changes of the sugar concentrations. The response time of the sensor fiber was about 4–5 min. The glucose sensor showed a good measurable fluorescence signal over a period of 11 days. The use of this assay for antibody/antigen system is proposed.

Tagging Retrovirus Vectors with a Metal Binding Peptide and One-Step Purification by Immobilized Metal Affinity Chromatography

ABSTRACT Retroviral vectors produced from packaging cells are invariably contaminated by protein, nucleic acid, and other substances introduced in the manufacturing process. Elimination of these contaminants from retroviral vector preparations is helpful to reduce unwanted side effects, and purified vector preparations are desirable to improve reproducibility of therapeutic effect. Here we report a novel approach to engineer a metal binding peptide (MBP)-tagged murine leukemia virus (MuLV), allowing for one-step purification of retroviral vectors by immobilized metal affinity chromatography (IMAC). We inserted a His6 peptide into an ecotropic envelope protein (Env) by replacing part of its hypervariable region sequence with a sequence encoding the His6 peptide. Display of the His6 tag on the surface of Env endowed the vectors with a high affinity for immobilized metal ions, such as nickel. We demonstrated that the His6-tagged MuLV could be produced to high titers and could be highly purified by one-step IMAC. The protein and DNA contaminants in the purified vector supernatants were below 7 μg/ml and 25 pg/ml, respectively, indicating a 1,229-fold reduction in protein contaminant level and a 6,800-fold reduction in DNA contaminant level. About 56% of the viral vectors were recovered in the IMAC purification. The purified vectors retained their functionality and infectivity. These results establish that an MBP can be functionally displayed on the surface of ecotropic retroviruses without interfering with their integrity, and MBP-tagged retroviral vectors can be highly purified by one-step IMAC.

THE PERMEABILITY OF GASES THROUGH REACTING SOLUTIONS: THE CARBON DIOXIDE-BICARBONATE MEMBRANE SYSTEM

The transport of a gas across a stationary hquld film containing reactive species 1s investigated for the purpose of determining gas permeablhtles or mass transfer coefficients m reacting solutions Under hnutmg condltlons when the reaction time constant far exceeds the dtiuslonal time constant, the flux of the transported gas follows Flcks law of dlffuslon Analytical series solution for the contnbutlon of the chemical reaction to the transport process 1s obtained usmg the technique of perturbation analysis, criteria for the validity of various terms m the series solution are presented The permeablhty of carbon dioxide m water and m 1N NaHCOrNa2C0, solution 1s estimated It 1s shown that a hleh degree of accuracy m the data 1s necessary for obtammg separate estimates of dlffuslvlty and sol&htyby this technique

MASS TRANSFER OF CO2 ACROSS MEMBRANES: FACILITATION IN THE PRESENCE OF BICARBONATE ION AND THE ENZYME CARBONIC ANHYDRASE

SUMMARY A theoretical and experimental analysis of facilitated transport of CO2 across membranes containing NaHCO3 and the enzyme carbonic anhydrase (carbonate hydro-lyase EC 4.2.1.1) is presented. The necessary diffusion reaction equations are derived and the system constraints defined. For the CO2-HCO3- system, mathematical simplifications based on the magnitude of various reaction and concentration terms are made to make the equations tractable to solution. The resultant equations are solved by a number of analytical and numerical techniques, each having a limited, though useful, range of validity. The experimental arrangement consists of a liquid membrane (created by soaking a porous filter paper in the test solution), a diffusion chamber, and gas metering and analysis equipment. Conditions were selected to cover the range from diffusionto reaction-dominated behavior. The flux of CO2 across a membrane containing ! M NaHCO3 was measured at various partial pressures of CO2 (2-28 in Hg) and with membrane thicknesses of 0.02, 0.06 and 0.10 cm. The extent of facilitation (defined as the ratio of reactionrelated flux to the expected Ficks Law flux in the absence of reaction) ranged from near zero to nearly 5 in these experiments. The agreement between model calculations and experimental observation was found to be excellent over the entire range of neardiffusion to near-equilibrium behavior. In the presence of enzyme carbonic anhydrase (0.10 mg/ml, activity approx. 80%) and 1 M NaHCO3, the CO2 flux across a 0.02 cm membrane was 3-10-fold higher than the corresponding flux in the absence of enzyme. From experiments at various enzyme concentrations and membrane thicknesses, it appeared that the apparent CO2 reaction rate was directly proportional to the enzyme concentration. The model calculations for the enzyme-catalyzed reactions agreed with the experimental observations to within ± 10 %.

Detection of tumor markers based on extinction spectra of visible light passing through gold nanoholes

The authors demonstrate that nanometric holes in optically thin gold films on glass slides can be used as biochemical sensors based on extinction spectra of visible light passing through these holes. Binding and adsorption of biomolecules to the surfaces of gold and glass result in a redshift of the localized surface plasmon resonance peak in the extinction spectrum of the gold holes. Selective sensing of antigens such as cancer antigen 19-9 (of less than 1pg on an ∼0.1mm2 probing area) can be realized using this type of devices after functionalizing the gold and glass surfaces with bioreceptors.The authors demonstrate that nanometric holes in optically thin gold films on glass slides can be used as biochemical sensors based on extinction spectra of visible light passing through these holes. Binding and adsorption of biomolecules to the surfaces of gold and glass result in a redshift of the localized surface plasmon resonance peak in the extinction spectrum of the gold holes. Selective sensing of antigens such as cancer antigen 19-9 (of less than 1pg on an ∼0.1mm2 probing area) can be realized using this type of devices after functionalizing the gold and glass surfaces with bioreceptors.