Heparin is a linear heterogeneous polysaccharide that carries the strongest negative charges in the biological system due to its high degree of sulfate substitution. Heparin has been used as the dominant choice for anticoagulation during cardiac surgery because of its effectiveness, low cost, and easy removal. Developing highly sensitive and highly selective assays for monitoring heparin levels in blood is required during and after surgery. In previous studies, electrostatic interactions are exploited to recognize heparin and changes in light signal intensity are used to sense heparin. Whether electrostatic interactions are sufficient to ensure specific heparin sensing while other anion molecules demonstrate slight interference in blood samples can be a particularly contentious issue.
To avoid the limitations arising from heparin sensing by electrostatic interaction, we propose a novel active heparin sensing platform. AT III labeled with QD specifically binds to active heparin molecules and forms the QDs-AT III-heparin complex. The complexes are then induced to aggregate upon addition of cationic surfactant micelles. The QDs attach onto the surface of micelles because of the electrostatic attractions between heparin and micelles. QD aggregations are observed and recognized based on their spectral images, which are recorded using a single-molecule spectral imaging fluorescence microscope. The ratio of QD aggregation spots to all counted fluorescence spots is proportional to heparin concentration under optimal conditions. Active heparin concentrations are then quantified in aqueous solution and 1000-fold diluted human blood plasma. The method demonstrates the following advantages. (i) The affinity interaction of AT III and heparin is employed instead of electrostatic attractions to improve specific binding. CTAB was only used as a bridge to form the QD-ATIII-heparin complex aggregates. (ii) Active components of heparin are quantified instead of the overall amount because AT III binds to the pentasaccharide sequence, the active region of heparin. (iii) The single-particle-level sensor significantly decreases the detectable concentration of heparin to 0.1 nM. The complicated sample can then be diluted continuously until the background exerts a minimal effect on the measurement. (iv) Finally, the quantification of the sensor is not dependent on the change in fluorescence intensity but on the aggregation ratio, which is helpful in minimizing the errors from the intensity fluctuation.
