Single-molecule force spectroscopy based atomic force spectroscopy, Fast Protein Liquid Chromatography (FPLC), Quartz Crystal Microbalance (QCM), and Electrochemical Impedance Spectroscopy (EIS), Two-Photon Polymerization (2PP), Scanning Electron Microscopy (SEM), Confocal Laser Microscopy (CLMS), Enzyme immunoassays, Dynamic Light Scattering (DLS), Circular dichroism (CD) spectroscopy, Surface Plasmon Resonance (SPR), Enzyme Immunoassays (EIA), Flow Cytometry (FC), Microfluidics, etc.
- Synthesis and characterization of magnetic nanoparticles
Platelets are transfused to prevent bleeding or to treat bacterial infection in many patients. To distinguish transfused platelets from the patient’s, own after transfusions, platelets are labeled with magnetic nanoparticles. Platelet labeling efficiency is enhanced when particles are conjugated with proteins and binding pathways of particles during platelet labeling have been determined (ACS Appl. Mater. Interfaces 2018, 10, 34, 28314-28321). However, the large variety of binding forces between particle and platelet and the aggregation of particles were still observed. This limitation may be due to the original characteristics of magnetic nanoparticles such as their shape/size, morphology and also the type of proteins conjugated on the surface of the particles.
Development of magnetic nanoparticles.
Iron oxide core of different shapes such as sphere and cubic shapes are synthesized and conjugated with protein.
To further improve platelet labeling, we cooperate with Dr. Jörg Schemberg to synthesize magnetic nanoparticles with different shapes/morphologies and to narrow the size distribution of the nanoparticles. We conjugate magnetic nanoparticles with proteins and characterize them when they interact with human platelets.
- HIT antibodies – Cancer cells
Many cancer patients are treated with heparins. Patients with heparin treatment may develop HIT antibodies. However, little is known about the role of HIT antibodies in cancer patients. We aim to investigate the interaction of HIT antibodies with cancer cells. Insights into the binding of HIT antibodies to cancer cells may help us to better manage HIT in cancer patients.
Platelets bound to breast cancer cells (left)
cultured on a petri dish and (right) in fluid phase.
- Controlling platelet-surface activation
Platelets can adhere to almost all nonphysiological surfaces and are quickly activated. Modulation of platelet-surface activation is important for many medical applications. We recently found that the degree of platelet-surface activation could be reduced by coating surfaces with collagen-G (Scientific Reports, 6(1), 25402, July 2016) or when they are seed on laminin coated nano-groove patterns. However, weak activation is still observed on all investigated surfaces. To further reduce platelet-surface activation, we aim to produce nanopatterns and nanoscaftfolds of different shapes and sizes using various types of polymers and modify them before seeding platelets on.
Controlling platelet-surface activation.
Nanostructured surfaces are fabricated and modified with different materials before seeding platelets on.
- Detection of HIT antibodies
Current enzyme immunoassays based optical detection are widely used due to their high sensitivity and fast turnover rate but the accuracy of these techniques is just ~50%. As HIT antibodies are the central problem that leads to platelet aggregation and activation, a technique for better detection of these antibodies is urgently needed. A false result may conduct treatment for patients without containing pathogenic HIT antibodies that can result in serious consequences such as venous limb gangrene or fatal hemorrhage. Recently, we found that HIT antibodies bind strongly to platelet membrane (Blood (2017) 129 (26): 3498–3501), (ACS Nano. 2018 Dec 26;12(12):12030-12041) and binding characteristics of HIT antibodies depend on their surrounding environment (J Thromb Haemost. 2019 Jul;17(7):1113-1119), (J. Phys. Chem. B 2020, 124, 8, 1438-1443). Based on these findings, we aim in two directions to better detect pathogenic HIT antibodies:
PF4/Heparin complexes are covalently immobilized on Au-sensor. HIT antibodies are added into the chamber for interaction with PF4/H complexes. Binding of HIT antibodies will be detected by impedance signals.
Biosensor for detection of HIT antibodies
Example of impedance signals show for baseline (blue), 1st (red), 2nd (green) and 3rd (violet) concentration added.
PF4/heparin complexes are immobilized on a 96-well plate to allow binding of anti-PF4/heparin antibodies. They are subsequently detected by a secondary goat anti-human IgG antibody using a chromogenic substrate that yields a visible color change, indicating their binding to the coated PF4/heparin complexes. The optical density (OD) of the sample was measured at wavelength 450 nm.
PF4 EIA. (left) Schematic model for the detection of HIT antibodies in EIA. (right) Current PF4 EIA provides an overlap OD signal (red area) of ~50% between HIT (red line) and non-HIT (black line) antibodies.