Increasing Binding Efficiency via Reporter Shape and Flux in a Viral Nanoparticle Lateral-Flow Assay

To identify factors controlling the performance of reporter particles in a sensitive lateral-flow assay (LFA), we investigated the effect of the flux and shape of filamentous bacteriophage (phage) on the performance of phage LFAs. Phage of three different lengths and diameters were modified with biotin and AlexaFluor 555 as binding and read-out elements, respectively. The binding efficiencies of the functionalized phage were tested in a fibrous glass LFA membrane modified with avidin. The total binding rate, quantified using real-time particle counting and particle image velocimetry, decreased monotonically with the average bulk flux of phage through the membrane. At the pore scale, more phage bound in regions with faster local flow, confirming that both average and local flux increased binding. The number of bound phage increased with the aspect ratio of the phage and scaled with the phage surface area, consistent with a binding interaction controlled by the number of recognition elements on the surface. Together, these results indicate that increasing the likelihood that recognition elements on the surface of phage encounter the fibers enhances the assay binding efficiency and suggests one origin for the improved performance of nonspherical phage reporters

DataSheet_1_A novel technology for home monitoring of lupus nephritis that tracks the pathogenic urine biomarker ALCAM.docx

IntroductionThe gold standard for diagnosis of active lupus nephritis (ALN), a kidney biopsy, is invasive with attendant morbidity and cannot be serially repeated. Urinary ALCAM (uALCAM) has shown high diagnostic accuracy for renal pathology activity in ALN patients.MethodsLateral flow assays (LFA) for assaying uALCAM were engineered using persistent luminescent nanoparticles, read by a smartphone. The stability and reproducibility of the assembled LFA strips and freeze-dried conjugated nanoparticles were verified, as was analyte specificity.ResultsThe LFA tests for both un-normalized uALCAM (AUC=0.93) and urine normalizer (HVEM)-normalized uALCAM (AUC=0.91) exhibited excellent accuracies in distinguishing ALN from healthy controls. The accuracies for distinguishing ALN from all other lupus patients were 0.86 and 0.74, respectively.ConclusionPeriodic monitoring of uALCAM using this easy-to-use LFA test by the patient at home could potentially accelerate early detection of renal involvement or disease flares in lupus patients, and hence reduce morbidity and mortality.</p

Deconstruction of a Nutlin: Dissecting the Binding Determinants of a Potent Protein–Protein Interaction Inhibitor

Protein–protein interaction (PPI) systems represent a rich potential source of targets for drug discovery, but historically have proven to be difficult, particularly in the lead identification stage. Application of the fragment-based approach may help toward success with this target class. To provide an example toward understanding the potential issues associated with such an application, we have deconstructed one of the best established protein–protein inhibitors, the Nutlin series that inhibits the interaction between MDM2 and p53, into fragments, and surveyed the resulting binding properties using heteronuclear single quantum coherence nuclear magnetic resonance (HSQC NMR), surface plasmon resonance (SPR), and X-ray crystallography. We report the relative contributions toward binding affinity for each of the key substituents of the Nutlin molecule and show that this series could hypothetically have been discovered via a fragment approach. We find that the smallest fragment of Nutlin that retains binding accesses two subpockets of MDM2 and has a molecular weight at the high end of the range that normally defines fragments

Impact of Pore–Walls Ligand Assembly on the Biodegradation of Mesoporous Organosilica Nanoparticles for Controlled Drug Delivery

Porous materials with molecular-scale ordering have attracted major attention mainly because of the possibility to engineer their pores for selective applications. Periodic mesoporous organosilica is a class of hybrid materials where self-assembly of the organic linkers provides a crystal-like pore wall. However, unlike metal coordination, specific geometries cannot be predicted because of the competitive and dynamic nature of noncovalent interactions. Herein, we study the influence of competing noncovalent interactions in the pore walls on the biodegradation of organosilica frameworks for drug delivery application. These results support the importance of studying self-assembly patterns in hybrid frameworks to better engineer the next generation of dynamic or “soft” porous materials

Metal Impurities Cause False Positives in High-Throughput Screening Campaigns

Organic impurities in compound libraries are known to often cause false-positive signals in screening campaigns for new leads, but organic impurities do not fully account for all false-positive results. We discovered inorganic impurities in our screening library that can also cause positive signals for a variety of targets and/or readout systems, including biochemical and biosensor assays. We investigated in depth the example of zinc for a specific project and in retrospect in various HTS screens at Roche and propose a straightforward counter screen using the chelator TPEN to rule out inhibition caused by zinc