Assessment of Shear-Dependent Kinetics of Primary Haemostasis With a Microfluidic Acoustic Biosensor.

Primary haemostasis is a complex dynamic process, which involves in-flow interactions between platelets and sub-endothelial matrix at the area of the damaged vessel wall. It results in a first haemostatic plug, which stops bleeding, before coagulation ensues and consolidates it. The diagnosis of primary haemostasis defect would benefit from evaluation of the whole sequence of mechanisms involved in platelet plug formation in flow. This work proposes a new approach that is based on characterization of the shear-dependent kinetics that enables the evaluation of the early stages of primary haemostasis. We used a label-free method with a quartz crystal microbalance (QCM) biosensor to measure the platelet deposits over time onto covalently immobilized type I fibrillar collagen. We defined three metrics: total frequency shift, lag time, and growth rate. The measurement was completed at four predefined shear rates prevailing in small vessels (500, 770, 1000 and 1500 s-1) during five minutes of perfusion with anticoagulated normal whole blood. The rate of the frequency shift over the first five minutes was strongly influenced by shear rate conditions, presenting a maximum around 770 s-1, and varying by a factor larger than three in the studied shear rate range. To validate the biosensor signal, the total frequency shift was compared to results obtained by atomic force microscopy (AFM) on final platelet deposits. The results show that shear-dependent kinetic assays are promising as an advanced method for screening of primary haemostasis.

Topology Challenge for the Assessment of Living Cell Deposits with Shear Bulk Acoustic Biosensor.

Shear bulk acoustic type of resonant biosensors, such as the quartz crystal microbalance (QCM), give access to label-free in-liquid analysis of surface interactions. The general understanding of the sensing principles was inherited from past developments in biofilms measurements and applied to cells while keeping the same basic assumptions. Thus, the biosensor readouts are still quite often described using 'mass' related terminology. This contribution aims to show that assessment of cell deposits with acoustic biosensors requires a deep understanding of the sensor transduction mechanism. More specifically, the cell deposits should be considered as a structured viscoelastic load and the sensor response depends on both material and topological parameters of the deposits. This shifts the paradigm of acoustic biosensor away from the classical mass loading perspective. As a proof of the concept, we recorded QCM frequency shifts caused by blood platelet deposits on a collagen surface under different rheological conditions and observed the final deposit shape with atomic force microscopy (AFM). The results vividly demonstrate that the frequency shift is highly impacted by the platelet topology on the bio-interface. We support our findings with numerical simulations of viscoelastic unstructured and structured loads in liquid. Both experimental and theoretical studies underline the complexity behind the frequency shift interpretation when acoustic biosensing is used with cell deposits.