5 research outputs found
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