Ralph Ballerstadt

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.

KINETICS OF DISSOLUTION OF CONCANAVALIN A/DEXTRAN SOLS IN RESPONSE TO GLUCOSE MEASURED BY SURFACE PLASMON RESONANCE

Abstract The dissolution of highly viscous dispersions composed of complexes of Concanavalin A (Con A) and high molecular-weight dextran (2000 kDa) in response to glucose was studied by using surface plasmon resonance (SPR). The viscous property of Con A/dextran sols is based on intermolecular affinity bonds between dextran and Con A. These receptor/ligand cross-linkages can be reversibly cleaved by the competing analyte glucose, resulting in a sol with a strongly reduced viscosity. We tested the concept of using the kinetics of sol dissolution as an analytical tool for glucose determination. Under this premise, a thin layer of the Con A/dextran dispersion was placed on the gold surface of a novel SPR-probe. The sols dissolution, when placed in contact with bulk solutions of various glucose concentrations, was monitored by measuring the shift of resonance angle. An increased rate of sol dissolution at increasing levels of glucose concentration was found, presumably due to a decrease of the sol viscosity which led to a faster diffusion and mass transport of the sol components into the bulk solution. The thickness of the sol layer turned out to be the rate-limiting step of the dissolution kinetics. Other factors such as sugar specificity and temperature had a significant affect on the kinetics of sol dissolution also. However, matrix effects of the test solution effected the overall kinetics of the solution only to a minimal extent, rendering the method interesting for biosensing purposes.

Sensor methods for use with microdialysis and ultrafiltration

Abstract The use of biosensors for metabolite and drug monitoring utilizing different micro dialysis sampling techniques is reviewed. After a brief introduction in the methods of microdialysis and ultrafiltration, general parameters that characterize the response of micro sensor devices (sensitivity range, sample requirements, response time) and the efficiency of micro sampling (charge and osmotic effects) are discussed. A variety of practical examples of biosensors used in micro sampling techniques are described. It is concluded that the intrinsic properties of biosensors are well suited for microdialysate monitoring, and that the progress in sensor technologies will open up the possibility of the use of integrated biosensor/microdialysis probe systems.

Development of a percutaneous fiberoptic hepatic venous localization catheter.

OBJECTIVE To develop a liver-specific biosensor system/catheter assembly that can be used to localize and cannulate the hepatic venous system without the need for fluoroscopic imaging. This would permit the bedside placement of a hepatic venous catheter for monitoring purposes without radiographic guidance. DESIGN Experimental, in vitro. STUDY SETTING Experimental laboratory at a university center. SUBJECT This was a simulation study to evaluate the ability of a cardiovascular monitoring catheter mounted with a liver-specific biosensor to anatomically identify a side arm tributary. The experimental system used for this study mimics the hepatic vein draining into the inferior vena cava and allows its localization without the need for assisted imaging. The biosensor design and catheter/sensor assembly function were studied in this in vitro model. INTERVENTIONS A liver-specific biosensor was developed by housing a homogeneous affinity fluorescence assay system sensitive to galactose in a microdialysis hollow fiber receptacle. A polyvinyl chloride tube containing a side arm was constructed to mimic the confluence of a venous tributary (i.e., the hepatic vein) with a major vascular channel (i.e., the vena cava). In this simulation, the side arm was continuously perfused with a liver-sensitive analyte (galactose) and the main channel was perfused with galactose-free buffer. A cardiovascular catheter containing a fiberoptic waveguide mounted with a galactose-sensitive fluorescent probe was advanced along the main conduit to assess its ability to identify the location of the galactose side arm infusion site. MEASUREMENTS AND MAIN RESULTS The response of the fiberoptic sensor to different galactose concentrations was assessed and found to be almost linear over the concentration range of 0 to 2 mM, which encompasses the expected utilization range of this system. The variability in identifying the galactose infusion point (simulated hepatic vein) in a 15-cm conduit was 1.7 to 2.8 mm, or 1.1% to 1.9%. CONCLUSIONS The construction of a catheter/sensor system with the ability to provide accurate spatial/anatomical localization data for the hepatic venous system is feasible. This assembly will eliminate the need for ancillary imaging systems for catheter/sensor delivery to an individual organ system and potentially can be positioned at the bedside in a fashion similar to the pulmonary artery flotation catheter.

A homogeneous affinity fluorescence assay system for galactose monitoring

Abstract Since the systemic venous-hepatic venous galactose gradient is normally quite large, the ability to detect different blood galactose concentrations in vivo would provide a mechanism for anatomic localization of the hepatic venous system. Furthermore, the development of a catheter assembly mounted with a galactose-sensitive biosensor would provide a technique for cannulating this organ system entirely using sensor guidance. In this report we describe a homogeneous affinity fluorescence assay system which can be contained in a dialysis hollow fiber for continuous galactose monitoring. The principle of this assay is based upon competition of freely permeable galactose for the specific binding interactions between a fluorescently labeled polysaccharide and lectin reagent pair. This competetive energy-transfer assay exhibits good sensitivity over a physiologically relevant galactose concentration range (0–2 mM), an acceptable response time (

Specular reflection vs. backscattering for transdermal OCT-based glucose monitoring with an implantable sensor system

The reliability of optical techniques for non-invasive monitoring of glucose can be significantly improved by the deployment of a subcutaneous implantable sensor that can closely track the changes in the local concentration of glucose in skin. We have developed a novel implantable sensor that can track glucose-induced changes in the optical turbidity of the implant. In this sensor, optical turbidity decreases significantly with increased glucose concentrations. We performed comparative measurements by optical coherence tomography (OCT) used to monitor backscattering or specular reflection originated from specific structures within the sensor and by collimated light transmission measurement technique to measure the changes in light attenuation as function of glucose concentration within the sensor as well as when the sensor was implanted in phantom media or in tissue samples. These measurements showed that glucose-induced changes in the transmission values derived from OCT monitoring of the sensor turbidity differed up two times from those obtained by collimated transparency measurement (CTM) technique. These results were used to determine the values for scattering coefficients of tissue and the sensor and to estimate the relative loss in sensor sensitivity as a function of implantation depth in tissue. The results suggest that the implantable sensor can be placed in turbid medium such as skin up to an optical depth of 12 mean free paths (mfp), one could expect. For a turbid medium such as skin with a scattering coefficient (µs ) of 10mm-1, this would result in geometrical depth of implantation at 1.2 mm beneath the tissue where sensor sensitivity of 50% or higher is expected. The study demonstrates that it could be feasible to engineer a novel optical sensor for glucose monitoring that can be implanted under the skin while providing a high degree of sensitivity and specificity for non-invasive glucose monitoring.

Prototype for an organ system (liver) localization cardiovascular catheter

This study describes the function and potential clinical utility of a sensor which can serve as a guidance mechanism allowing for the selective cannulation of the hepatic venous system without the need for additional imaging technology. The sensor is based upon a homogeneous affinity fluorescence assay system utilizing the lectin Ricinus cummunis agglutinin I and covalently linked to the fluorophol Alexa 488 and its conjugate polydextran labeled with rhodamine and lactose. The affinity between these two macromolecules is sensitive to ambient galactose concentration which exists as a steep gradient at the hepatic venous/vena caval junction allowing this anatomic region to be discriminated from irrelevant regions. This sensor system permits venous access for additional monitoring approaches such as venous oximetry.