Frank K. Gehring

Blood Coagulation Thromboplastine Time Measurements on a Nanoparticle Coated Quartz Crystal Microbalance Biosensor in Excellent Agreement with Standard Clinical Methods

Measurements of parameters of hemostasis like thromboplastine time (PT) have primary significance in many clinical settings including extensive surgery, dialysis or innate disorders of hemostasis. Recently, several reports have documented the principle suitability of Quartz Crystal Microbalances (QCM) for measuring parameters of hemostasis like PT or platelet aggregation. But for the establishment of an exact QCM based method as alternative to standard coagulometer measuring QCM coatings with significantly enhanced robustness and reusability have to be worked out. For this purpose we utilized a new semi-automated equipment qCell T for measuring and compared different coatings consisting of polymer films and absorbed nanoparticles (NPs). We demonstrated that affinity based poly ethylene NPs absorbed to polymer films on the QCM constitute a powerful tool with no need for pretreatment for measuring PT in whole blood samples in real time, while these coatings are reusable up to 10 times. PT measurements in excellent agreement with coagulometer tests pave the way for possible future application of QCM in clinical routine.

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.

NCO-sP(EO-stat-PO) Coatings on Gold Sensors—a QCM Study of Hemocompatibility

The reliability of implantable blood sensors is often hampered by unspecific adsorption of plasma proteins and blood cells. This not only leads to a loss of sensor signal over time, but can also result in undesired host vs. graft reactions. Within this study we evaluated the hemocompatibility of isocyanate conjugated star shaped polytheylene oxide—polypropylene oxide co-polymers NCO-sP(EO-stat-PO) when applied to gold surfaces as an auspicious coating material for gold sputtered blood contacting sensors. Quartz crystal microbalance (QCM) sensors were coated with ultrathin NCO-sP(EO-stat-PO) films and compared with uncoated gold sensors. Protein resistance was assessed by QCM measurements with fibrinogen solution and platelet poor plasma (PPP), followed by quantification of fibrinogen adsorption. Hemocompatibility was tested by incubation with human platelet rich plasma (PRP). Thrombin antithrombin-III complex (TAT), β-thromboglobulin (β-TG) and platelet factor 4 (PF4) were used as coagulation activation markers. Furthermore, scanning electron microscopy (SEM) was used to visualize platelet adhesion to the sensor surfaces. Compared to uncoated gold sensors, NCO-sP(EO-stat-PO) coated sensors revealed significant better resistance against protein adsorption, lower TAT generation and a lower amount of adherent platelets. Moreover, coating with ultrathin NCO-sP(EO-stat-PO) films creates a cell resistant hemocompatible surface on gold that increases the chance of prolonged sensor functionality and can easily be modified with specific receptor molecules.

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

BackgroundAn 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.MethodsBinding 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.ResultsFrequency 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.ConclusionsIn 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.

Quarzkristall-Mikrowaagen-Technologie als neue bioanalytische Plattform

QCM-D is a surface-sensitive technique for real-time monitoring of biolayers on surfaces with regard to adsorption and desorption, molecular interactions and structural properties. To date, this old-established technique is employed for studies of cellular processes, both, in nanotechnology and cell biology. Interactions among molecules, cells or between cells and the environment are observable in their natural unaltered states. QCM-D thus provides a unique insight into the complex world of biology.