Nanomechanical resonators for clinical coagulation diagnostics and malaria vaccine candidates analysis

Abstract

The development of a microcantilevers-based (MC-based) clinical diagnostic instrument that employs the dynamic mode of operation (microresonators) is presented. The device exploits microresonator arrays that are actively driven within a specific frequency bandwidth by a piezo-electric element. The microresonators are microcantilevers structures clamped at one end. We successfully applied the device for clinical coagulation diagnostics and malaria vaccine candidates testing. First of all, we designed and developed a new thermoregulated enclosure that allows experiments at a stable temperature ranging from room temperature to 37°C. The temperature stability is ca. 0.015°C. Next, the microfluidics system was tested and optimal parameters for uniform mixing of fluids were determined. The uniform mixing is challenging in small volumes but is mandatory for repeatable blood coagulation measurements. Indeed, clinical guidelines recommend specific concentrations of reagents that have to be uniformly mixed with the patients' plasma respecting specific timings. In order to measure coagulation parameters, we employed microresonators in dynamic mode. The resonant frequency and quality factor shifts are correlated to viscosity and density changes in the liquid surrounding the oscillating structures. We improved the evaluation of the phase signal by introducing a third signal shifted by 90° (Hilbert transform). The third signal allows evaluation of the phase in the full 360° circle. Next, we presented a rigorous theoretical model that computes the density and viscosity of the liquid starting from the measurement of resonant frequency and quality factor. The newly developed device was tested with two biological applications: clinical coagulation diagnostics and malaria vaccine candidates testing. In the field of coagulation diagnostics, we accurately measured clinical coagulation times such as prothrombin time (PT) and activated partial thromboplastin time (aPTT), in human blood plasma, respecting clinical guidelines that demand few seconds’ sensitivity. We then diagnosed a specific factor deficiency (factor IX, hemophilia B) through an in-situ integrated immunoassay. Along with the PT or the aPTT times, the assay also measures the specific factor deficiency and provides quantitative parameters such as fibrin polymerization rate and subsequent crosslinking rate before reaching saturation (final clot strength). Finally, we measured the activity of an impaired fibrinolysis system and the effects of the drug heparin, a common thrombosis therapeutic drug. As expected, heparin prolongs the coagulation time, but does not interfere with the final clot strength. In the field of malaria vaccine candidates testing, we measured the antigenic properties of three different vaccine candidates. We showed that by immobilizing antibodies on the sensors surfaces we can detect the specific interaction antibody-antigen in a quantitative label-free manner. Finally, we measured the immune response of immunized mice and human volunteers. The assay represents a clear advancement in the field of immunological methods and blood coagulation diagnostics. The small sensors size and the measurement chamber volume (6 ul) opens up possibilities for great miniaturization and subsequent point-of-care (POC) device development. The differential readout of cantilever arrays measurements provides additional possibilities to distinguish sequence differences in antigen epitopes. Such a measurement is currently not possible with ELISA immunoassay.