Real-time measurement of Plasmodium falciparum-infected erythrocyte cytoadhesion with a quartz crystal microbalance.

BACKGROUND: An important virulence mechanism of the malaria parasite Plasmodium falciparum is cytoadhesion, the binding of infected erythrocytes to endothelial cells in the second half of asexual blood stage development. Conventional methods to investigate adhesion of infected erythrocytes are mostly performed under static conditions, many are based on manual or semi-automated read-outs and are, therefore, difficult to standardize. Quartz crystal microbalances (QCM) are sensitive to nanogram-scale changes in mass and biomechanical properties and are increasingly used in biomedical research. Here, the ability of QCM is explored to measure binding of P. falciparum-infected erythrocytes to two receptors: CD36 and chondroitin sulfate A (CSA) under flow conditions. METHODS: Binding of late stage P. falciparum parasites is measured in comparison to uninfected erythrocytes to CD36- and CSA-coated quartzes by QCM observing frequency shifts. CD36-expressing cell membrane fragments and CSA polysaccharide were coated via poly-L-lysine to the quartz. The method was validated by microscopic counting of attached parasites and of erythrocytes to the coated quartzes. RESULTS: Frequency shifts indicating binding of infected erythrocytes could be observed for both receptors CD36 and CSA. The frequency shifts seen for infected and uninfected erythrocytes were strongly correlated to the microscopically counted numbers of attached cells. CONCLUSIONS: In this proof-of-concept experiment it is shown that QCM is a promising tool to measure binding kinetics and specificity of ligand-receptor interactions using viable, parasite-infected erythrocytes. The method can improve the understanding of the virulence of P. falciparum and might be used to cross-validate other methods.

Utilisation of Quartz Crystal Microbalance Sensors with Dissipation (QCM-D) for a Clauss Fibrinogen Assay in Comparison with Common Coagulation Reference Methods.

The determination of fibrinogen levels is one of the most important coagulation measurements in medicine. It plays a crucial part in diagnostic and therapeutic decisions, often associated with time-critical conditions. The commonly used measurement is the Clauss fibrinogen assay (CFA) where plasma is activated by thrombin reagent and which is conducted by mechanical/turbidimetric devices. As quartz crystal microbalance sensors with dissipation (QCM-D) based devices have a small footprint, can be operated easily and allow measurements independently from sample transportation time, laboratory location, availability and opening hours, they offer a great opportunity to complement laboratory CFA measurements. Therefore, the objective of the work was to (1) transfer the CFA to the QCM-D method; (2) develop an easy, time- and cost-effective procedure and (3) compare the results with references. Different sensor coatings (donor's own plasma; gold surface) and different QCM-D parameters (frequency signal shift; its calculated turning point; dissipation signal shift) were sampled. The results demonstrate the suitability for a QCM-D-based CFA in physiological fibrinogen ranges. Results were obtained in less than 1 min and in very good agreement with a standardized reference (Merlin coagulometer). The results provide a good basis for further investigation and pave the way to a possible application of QCM-D in clinical and non-clinical routine in the medical field.

Investigation of prothrombin time in human whole-blood samples with a quartz crystal biosensor.

Monitoring of blood coagulation and fibrinolysis is an important issue in treatment of patients with cardiovascular problems and in surgery when blood gets into contact with artificial surfaces. In this work a new method for measuring the coagulation time (prothrombin time, PT) of human whole-blood samples based on a quartz crystal microbalance (QCM) biosensor is presented. The 10 MHz sensors used in this work respond with a frequency shift to changes in viscosity during blood clot formation. For driving and for readout of the quartz, both a network analyzer and an oscillator circuit were utilized. The sensor surfaces were specifically coated with a thin polyethylene layer. We found that both frequency analysis methods are suitable to measure exact prothrombin times in a very good conformity with a mechanical coagulometer as a reference. The anticoagulant effect of heparin on the prothrombin time was exemplarily shown as well as the reverse effect of the heparin antagonist polybrene. The change of the viscoelastic properties during blood coagulation, reflected by the ratio of frequency and dissipation shifts, is discussed for different dilutions of the whole-blood samples. In conclusion, QCM is a distinguished biosensor technique to determine prothrombin time and to monitor heparin therapy in whole-blood samples. Due to the excellent potential of miniaturization and the availability of direct digital signals, the method is predestinated for incorporation and integration into other devices and is thus opening the field of application for inline coagulation diagnostic in extracorporeal blood circuits.

Platelet aggregation monitoring with a newly developed quartz crystal microbalance system as an alternative to optical platelet aggregometry.

The objective of this study was to establish a new test system for the monitoring of platelet aggregation during extracorporeal circulation (ECC) procedures. Even though extensive progress has been made in improving the haemocompatibility of extracorporeal circulation devices, activation of blood coagulation, blood platelets and inflammatory responses are still undesired outcomes of cardiopulmonary bypass. This study deals with an approach towards a platelet aggregation measuring system using a newly developed quartz crystal microbalance (QCM) system. Since QCM is a rarely used technique in the field of blood analytics, the challenge was to transfer the well established methods of aggregometry to the new test system. In a QCM system, either bare gold or fibrinogen-coated sensors were incubated with ADP or arachidonic acid (AA) stimulated platelet rich plasma. For negative controls the GPIIb/IIIa inhibitory antibody abciximab (Reopro®) was used as an inhibitor of platelet aggregation. During incubation, the frequency shifts of the sensors were recorded. The results gained from the QCM system were compared to results gained by optical platelet aggregometry (born aggregometry). For additional visualization of platelet adhesion to the sensor surfaces, fluorescent microscopy and scanning electron microscopy were used. The QCM sensor was able to detect platelet aggregation in both uncoated and fibrinogen coated sensors. The measuring curves of aggregation measurements and controls were clearly distinguishable from each other in terms of frequency shifts and kinetics. For aggregation measurements and inhibited controls the therapeutic diagnosis of platelet function is identical between aggregometer and QCM data. In future, QCM based measuring devices may become an alternative to established point of care methods for rapid bedside testing of platelet aggregation.

Coordination Chemistry of the Carboxylate Type Siderophore Rhizoferrin: The Iron(III) Complex and Its Metal Analogs.

Rhizoferrin is a member of a new class of siderophores (microbial iron transport compounds) based on carboxylate and hydroxy donor groups rather than the commonly encountered hydroxamates and catecholates. We have studied the coordination chemistry of rhizoferrin (Rf), as a representative of this group, with Fe(3+), Rh(3+), Cr(3+), Al(3+), Ga(3+), VO(2+), and Cu(2+). The metal complexes have been studied by UV-vis, CD, NMR, and EPR spectroscopies and mass spectrometry. The formation constants for the iron complex have also been measured and yield a log K(LFe) of 25.3. The Rh and Cr rhizoferrin complexes are unusual in that they appear to adopt a chirality about the metal center that is the opposite of the native iron analog. Several of the alternative metal ion complexes are found to have biological activity toward Morganella morganii in a plate type assay.

The configuration of the chiral carbon atoms in staphyloferrin A and analysis of the transport properties in Staphylococcus aureus.

Staphyloferrin A, the iron-transporting siderophore of Staphylococci, contains two citric acid residues linked to a D-ornithine backbone, having thus three chiral centers. While the chirality of the backbone can be determined after hydrolysis, the chirality of the two citryl residues can only be determined from the intact staphyloferrin A molecule by circular dichroism spectra. The chirality of the quarternary carbon atoms of citryl residues in fungal rhizoferrin and bacterial enantio-rhizoferrin have been determined previously to be R,R and S,S respectively. The present investigation shows that of the three chiral centers in staphyloferrin A, the citryl residues can be assigned an S,S-configuration by comparison with synthetic analogs, confirming a common chirality among the bacterial enantio-rhizoferrin and staphyloferrin A. This suggests that the bacterial carboxylates originate from a common biosynthetic pathway leading to an S,S-configuration, while the fungal rhizoferrin possessing an R,R-configuration must have a different biosynthetic origin. Growth promotion tests with staphylococci revealed that the S,S-configuration of staphyloferrin A and enantio-rhizoferrin enabled iron uptake, while the fungal rhizoferrin with R,R-configuration was not utilized.