A Point-of-Care Quartz Biosensor Chip for Detection of PF4/Heparin Complexes

Abstract

BACKGROUNDOur recent studies indicate that circulating PF4/heparin complexes activate complement and contribute to a subsequent immune response seen in heparin-induced thrombocytopenia (HIT) (Khandelwal, Blood 2016). We undertook studies to develop a sensitive assay for measuring circulating PF4/heparin. To do so, we utilize a quartz crystal microbalance (QCM) device, which is a piezoelectric acoustic resonator that detects decreases in the resonance frequency of the quartz crystal due to the increased mass caused by molecules binding to the electrode surface. We hypothesize that a novel label-free QCM biosensor functionalized with PF4/heparin capture antibodies can provide for robust quantitative antigen detection and physical characterization of antigen-antibody interactions. METHODSWe formed a mixed self-assembled monolayer by using thiol-functionalized polyethylene glycols (PEGs) with -OH and biotin terminated head-groups on gold electrodes of quartz biosensor (Nelson; Langmuir 2001). PEG-backfilling of this surface provided protein non-fouling effects (1% BSA adsorbed only 10 ng/cm2), while biotin end-groups enabled close to full surface coverage of streptavidin (180 ng/cm2). This surface became our platform to immobilize biotinylated KKO (anti-PF4/heparin IgG2Κb monoclonal antibody, developed in Arepally Lab) and TRA (isotype IgG2Κb control antibody; developed in Arepally Lab).To characterize the sensor's detection ability, we flowed recombinant PF4 (provided by M. Poncz, Children's Hospital of Philadelphia) with a range of heparin (Elkins-Sinn, NJ) concentrations. The solutions are introduced to the biosensors 10 minutes after mixing the heparin and flowed through the system until the frequency readout stabilized. We determined the amount of captured PF4/heparin complexes and the layer thickness by monitoring the sensor's resonance frequency shifts and acoustic dissipation.The surface distribution of PF4/heparin complexes on KKO-functionalized surfaces was imaged by tapping-mode atomic force microscopy (AFM). The imaging results are used to verify layer thickness calculated by acoustic modeling of the QCM signal. RESULTSIn initial experiments, KKO showed no changes in the presence of buffer or PF4 alone. However, when PF4 (10 μg/ml) was infused with increasing amounts of heparin (0.2, 1 or 10 U/ml) significant binding was detected starting at 0.2 U/ml heparin (Figure A). The acoustic modeling of the KKO-bound PF4 complexes with 0.2 U/ml heparin suggested that the macromolecular complexes were nearly 25 nm high. These results are consistent with the neutralization model for the role of PF4 in heparin-induced thrombocytopenia, in which the amount of PF4/ heparin complexes peaks at a specific molar ratio of PF4:heparin (Kowalska; Thrombosis Research 2010). Comparatively, almost no binding occurred when the quartz crystal was functionalized with TRA. This result not only demonstrates the protein non-fouling capacity of the PEG backfilling of the surface, but also shows the specificity of the surface-immobilized assay.To gauge the sensitivity of the QCM biosensor to lower concentrations of PF4 and heparin that may be found in vivo, we tested 1µg/mL PF4 with lower heparin concentrations (0.02-0.05 U/ml). As shown in Figure B, a nearly linear KKO binding response was observed in this range capable of distinguishing heparin concentrations that differed by as little as 0.01 U/ml. The highest adsorption was observed at heparin concentration of 0.02 U/ml with associated atomic force microscopy (AFM) images again showing spherical particles with 25 nm diameter on the KKO immobilized surface. CONCLUSIONSHighly specific binding to PF4 complexes demonstrates the sensitivity of the QCM platform and its ability to distinguish differences between heparin concentrations of even 0.01 U/ml. Since QCM is a mass-sensing biosensor, it has a unique sensitivity advantage while sensing high molecular weight macromolecular complexes. This, combined with possible miniaturization of the sensor and integration with lab-on-a-chip systems, creates the potential for point-of-care bed-side detection of PF4/heparin. [Display omitted] DisclosuresArepally:Biokit: Patents & Royalties.