Identification and characterisation of fluorescent substances in spent dialysis fluid.

Patients who suffer from end-stage renal disease require renal replacement therapy, including haemodialysis. While applying extracorporeal blood treatment, uraemic toxins accumulated in the patients' blood pass into a physiological solution, the dialysis fluid. Thus, important information about the patient's health status can be obtained by analysing the spent dialysis fluid. To make use of this information, corresponding analysis concepts must be developed. In this context, this article reports the analysis of fluorescence in spent dialysis fluid. Excitation and emission maxima of fluorescence in spent dialysis fluid were recorded, and the main fluorescent substances were identified and quantified using high-performance liquid chromatography analysis. Fluorescence in spent dialysis fluid has two prominent excitation maxima at λex1 = 228 nm and λex2 = 278 nm. However, both excitation maxima cause emission with maxima at λem = 350 nm. Identification of fluorescent substances using high-performance liquid chromatography showed that the main contributors to the overall fluorescence in spent dialysis fluid are tyrosine, tryptophan, indoxyl sulphate and indole-3-acetic acid. However, these substances are responsible for only one-third of the overall fluorescence of spent dialysis fluid. A large number of substances, each of which contributes only to a small part to the overall fluorescence, emit the remaining fluorescence.

A feedback system that combines monitoring of systolic blood pressure and relative blood volume in order to prevent hypotensive episodes during dialysis.

Hypotensive Episodes (HEs) are one of the most common complications during dialysis. Occurrence of HEs can be reduced by applying physiological closed loop systems that monitor physiological parameter(s) and adjust dialysis related parameter(s). We developed a physiological closed loop control system (PCLCS) that monitors systolic blood pressure (sysBP) and relative blood volume (RBV) and calculates the net fluid removal (nfr) rate during dialysis. The performance of PCLCS was compared in the laboratory to a feedback system that monitors only RBV (BVFS). A laboratory test setup was developed to test the feedback systems. The test setup simulates nfr-rate and refilling of a patient's intravascular fluid. We studied the impact of the feedback systems PCLCS and BVFS on the number of HEs (sysBP < 90 mmHg), on the variance of sysBP and RBV, on pre to post sysBP and RBV and on the achievement of the nfr-volume. PCLCS allowed 80% less HEs than BVFS (p < 0.001). Variance of sysBP and RBV were reduced by 41.8% and by 52% (p < 0.001), respectively, when using PCLCS. There were no differences between pre to post sysBP nor between pre to post RBV when comparing PCLCS to BVFS. The nfr-volume was achieved by both feedback systems.

Quantitative analysis of energy transfer between fluorescent proteins in CFP-GBP-YFP and its response to Ca2+.

This article reports the full characterisation of the optical properties of a biosynthesised protein consisting of fused cyan fluorescent protein, glucose binding protein and yellow fluorescent protein. The cyan and yellow fluorescent proteins act as donors and acceptors for intramolecular fluorescence resonance energy transfer. Absorption, fluorescence, excitation and fluorescence decays of the compound protein were measured and compared with those of free fluorescent proteins. Signatures of energy transfer were identified in the spectral intensities and fluorescence decays. A model describing the fluorescence properties including energy transfer in terms of rate equations is presented and all relevant parameters are extracted from the measurements. The compound protein changes conformation on binding with calcium ions. This is reflected in a change of energy transfer efficiency between the fluorescent proteins. We track the conformational change and the kinetics of the calcium binding reaction from fluorescence intensity and decay measurements and interpret the results in light of the rate equation model. This visualisation of change in protein conformation has the potential to serve as an analytical tool in the study of protein structure changes in real time, in the development of biosensor proteins and in characterizing protein-drug interactions.

Wireless implantable electronic platform for chronic fluorescent-based biosensors.

The development of a long-term wireless implantable biosensor based on fluorescence intensity measurement poses a number of technical challenges, ranging from biocompatibility to sensor stability over time. One of these challenges is the design of a power efficient and miniaturized electronics, enabling the biosensor to move from bench testing to long term validation, up to its final application in human beings. In this spirit, we present a wireless programmable electronic platform for implantable chronic monitoring of fluorescent-based autonomous biosensors. This system is able to achieve extremely low power operation with bidirectional telemetry, based on the IEEE802.15.4-2003 protocol, thus enabling over three-year battery lifetime and wireless networking of multiple sensors. During the performance of single fluorescent-based sensor measurements, the circuit drives a laser diode, for sensor excitation, and acquires the amplified signals from four different photodetectors. In vitro functionality was preliminarily tested for both glucose and calcium monitoring, simply by changing the analyte-binding protein of the biosensor. Electronics performance was assessed in terms of timing, power consumption, tissue exposure to electromagnetic fields, and in vivo wireless connectivity. The final goal of the presented platform is to be integrated in a complete system for blood glucose level monitoring that may be implanted for at least one year under the skin of diabetic patients. Results reported in this paper may be applied to a wide variety of biosensors based on fluorescence intensity measurement.

A novel point of care diagnostic device: impedimetric detection of a biomarker in whole blood.

There is an unmet medical need for a more reliable and earlier assessment of patients suspected of having a deep vein thrombosis. We describe a novel approach which is developing a highly reliable, accurate, portable and handheld prototype medical diagnostic device to improve radically the speed, accuracy and reliability with which DVT and related blood clotting conditions can be assessed. The device will measure whole blood concentration of D-dimer, a recognized biomarker of increased blood clotting activity, and through innovation in the development of a novel detection, measurement and reporting system, will offer the opportunity to use the test in the point of care setting. The device combines innovation in antibody bio-engineering for high specificity immunoassay-based diagnostics and nano/micro engineered impedimetric analysis electrodes incorporating a biocompatible polymer substrate with development of a disposable microfluidic manifold specifically enabling diagnostics at the point-of-first-contact.