4830 Chem. Commun., 2013, 49, 4830--4832 This journal is c The Royal Society of Chemistry 2013 Cite this: Chem. Commun., 2013, 49, 4830 A simple new competition assay for heparin binding in serum applied to multivalent PAMAM dendrimers† Stephen M. Bromfield, a Paola Posocco, b Maurizio Fermeglia, b Sabrina Pricl, bc Julia ´n Rodrı ´guez-Lo ´pez d and David K. Smith* a We report a competition assay using our recently reported dye Mallard Blue, which allows us to identify synthetic heparin binders in competitive media, including human serum – using this we gain insight into the ability of PAMAM dendrimers to bind heparin, with the interesting result that low-generation G2-PAMAM is the preferred heparin binder. Heparin is the most charge-dense naturally occurring polyanion in biological systems (Fig. 1), 1 and is used as an anti-coagulant drug, for example during surgery. There has been a surge of interest in develop- ing heparin sensors, 2 and we recently reported a simple new sensor, Mallard Blue (MalB, Fig. 2) capable of ultra-high-affinity sensing of clinically-relevant heparin levels in serum. 3 In this paper, we demon- strate the use of MalB in a novel spectroscopic assay to probe the heparin binding ability of synthetic systems with potential applications for post-surgical heparin removal. Currently, the only licensed heparin reversal agent is protamine sulfate: a cationic arginine-rich protein of ill- defined structure and M r 3000–4500 Da derived from shellfish (Fig. 1). 4 Protamine causes adverse reactions in up to 10% of patients and 2.6% of cardiac surgeries experience serious complications. 5 There is there- fore interest in developing a replacement heparin reversal agent. 6 Previous approaches to screening heparin binders have used radio- isotopes, affinity co-electrophoresis, 7 competitive inhibition assays 8 and recently, turbidimetric screening. 9 Having previously demonstrated that MalB could detect heparin in competitive conditions, we designed a simple dye- displacement competition assay (Fig. 2) to probe synthetic heparin binders. Heparin was mixed with MalB and then the potential heparin binder was added. In the presence of increasing amounts of the heparin binder, MalB should be displaced from heparin and the absorbance intensity should increase. This assay can be used to calculate binding parameters. The EC 50 (effective concentration) is the concentration of binder required to displace 50% of MalB from heparin. The CE 50 (charge excess) is the number of positive charges required per heparin negative charge to achieve 50% displacement of MalB. The data can also be reported in terms of the mass of binder required to bind 100 international units of heparin – the clinical standard. To test the assay, we studied PAMAM (poly(amidoamine)) dendrimers with different generations of branching. PAMAMs (see ESI†), first reported by Tomalia and co-workers, 10 which exist as monodisperse individual nanoscale units in water, are known heparin binders. 11 Dendrimers can mimic many aspects of protein behaviour, 12 and we hoped that by assaying PAMAMs and benchmarking the data Fig. 1 Major heparin disaccharide repeat unit (left) and an example protamine (right, box). 7 Fig. 2 Schematic of competition assay using MalB. MalB binds to heparin but on addition of a competitive heparin binder, MalB is released. a Department of Chemistry, University of York, Heslington, York, YO10 5DD, UK. E-mail: david.smith@york.ac.uk b Molecular Simulation Engineering (MOSE) Laboratory, Department of Engineering and Architecture (DEA), University of Trieste, 34127 Trieste, Italy c National Interuniversity Consortium for Material Science and Technology (INSTM), Research Unit MOSE-DEA, University of Trieste, 34127 Trieste, Italy d A ´ rea de Quı ´mica Orga ´nica, Facultad de Ciencias y Tecnologı ´as Quı ´micas, Universidad de Castilla la Mancha, 13071 Ciudad Real, Spain † Electronic supplementary information (ESI) available: Assay conditions and data, further details of MD modelling. See DOI: 10.1039/c3cc41251b Received 17th February 2013, Accepted 11th April 2013 DOI: 10.1039/c3cc41251b www.rsc.org/chemcomm ChemComm COMMUNICATION Downloaded by University of York on 13/05/2013 17:31:46. Published on 18 April 2013 on http://pubs.rsc.org | doi:10.1039/C3CC41251B View Article Online View Journal | View Issue