Simple Fluidic System for Purifying and Concentrating Diagnostic Biomarkers Using Stimuli-Responsive Antibody Conjugates and Membranes

We report a simple fluidic system that can purify and concentrate diagnostic biomarkers through the capture and triggered release of stimuli-responsive polymer-antibody conjugates at porous membranes that are grafted with the same stimuli-responsive polymer. This technique is applied here to the capture and detection of a model streptavidin antigen and subsequently to clinical ranges of the malaria antigen Plasmodium falciparum histidine-rich protein 2 (PfHRP2) from spiked human plasma. The carboxyl end-groups of semi-telechelic poly(N-isopropylacrylamide) (pNIPAAm) synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization were modified with tetrafluorophenol to yield amine-reactive ester groups for conjugation to amine groups of anti-streptavidin and anti-PfHRP2 antibodies. Stimuli-responsive membranes were constructed from 1.2 mu m pore-size, hydroxylated, nylon-6,6 filters (Loprodyne, from Pall Corporation). The surface hydroxyl groups on the filters were conjugated to a 2-ethylsulfanylthiocarbonylsulfany1-2-methyl propionic acid (EMP) RAFT chain transfer agent, and the surface-grafted pNIPAAm was obtained by subsequent polymerization. The number average molecular weight (Mn) and polydispersity indices (PDI) of the surface grafts were characterized, and membranes with either 4100 and 8400 dalton pNIPAAm grafts showed greater than 80% anti-streptavidin capture efficiency. The 8400 dalton-graft membrane showed the highest release efficiency, and it was demonstrated that at 0.2 nM starting concentration the streptavidin could be concentrated approximately 40-fold by releasing into a small 50,mu L volume. This concentrator system was applied to the capture and concentration of the PfHRP2 antigen, and results showed that the PfHRP2 antigen could be processed and detected at clinically relevant concentrations of this malaria biomarker.