Digital enzyme-linked immunosorbent assays with sub-attomolar detection limits based on low numbers of capture beads combined with high efficiency bead analysis.

We report the development of digital enzyme-linked immunosorbent assays (ELISAs) based on single molecule arrays (Simoa) with improved sensitivities over conventional digital ELISA, enabling detection of proteins at sub-attomolar concentrations. The improvements in sensitivity were based on using fewer beads to capture the target proteins (≤5000 vs.∼500 000 beads) that increased the ratio of molecules to beads, and increasing the fraction of beads that were analyzed (bead read efficiency) from ∼5% to ∼50%. Bead read efficiency was increased by: a) improving the loading of beads into arrays of microwells by combining capillary and magnetic forces in a method called magnetic-meniscus sweeping (MMS); b) using a centrifugal washer to minimize bead loss during the assay; and, c) improved optics and image analysis to enable the analysis of more microwells. Using this approach, we developed an assay for IL-17A with a limit of detection (LOD) of 0.7 aM, 437-fold more sensitive than standard digital ELISA. A digital ELISA with improved sensitivity was used to measure IL-17A in 100 serum and plasma samples with 100% detectability, compared to 51% for standard digital ELISA. Low numbers of capture beads yielded improved LODs for IL-12p70 (0.092 aM), p24 (9.1 aM), and interferon alpha (45.9 aM). IL-4 and PSA showed no improvements in sensitivity using fewer beads, primarily due to low antibody loading on beads and increased non-specific binding, respectively. The results were consistent with a kinetic model of binding that showed that combining capture antibodies with high on-rates with high antibodies per bead yields the greatest improvement in sensitivity.

Metal ion-sensitive holographic sensors.

Holographic sensors for Na+ and K+ have been fabricated from crown ethers incorporated into polymeric hydrogels. The methacrylate esters of a homologous series of hydroxyether crown ethers were synthesized and copolymerized with hydroxyethyl methacrylate and the cross-linker ethylene dimethacrylate (3 mol %) to form stable hydrogel films (approximately 10 m thick) containing covalently bound (0-97 mol %) 12-crown-4, 15-crown-5, and 18-crown-6 pendant functionalities. The films were transformed into silver-based volume holograms using a diffusion method coupled with a holographic recording using a frequency-doubled Nd:YAG laser. The resulting holographic reflection spectrum was used to characterize the shrinkage and swelling behavior of the holograms as a function of polymer composition and the nature and concentration of alkali, alkaline earth, and NH4+ ions in the test media. Optimized film compositions containing 50 mol % crown ether showed substantial responses (< or = 200 nm) within 30 s at ion concentrations of < or = 30 mM, which could be rationalized on the basis of the known complexation behavior of the crown ethers. An 18-crown-6 holographic film was shown to be able to quantitate K+ concentrations over the physiologically relevant range. It was virtually unaffected by variations in the Na+ background concentration within the normal physiological variation (approximately 0.13-0.15 M) and shows promise for developing simple, low-cost K+ sensors for medical applications.