A Colloidal Stability Assay Suitable for High-Throughput Screening

A library of 32 polystyrene copolymer latexes, with diameters ranging between 53 and 387 nm, was used to develop and demonstrate a high-throughput assay using a 96-well microplate platform to measure critical coagulation concentrations, a measure of colloidal stability. The most robust assay involved an automated centrifugation–decantation step to remove latex aggregates before absorbance measurements, eliminating aggregate interference with optical measurements made through the base of the multiwell plates. For smaller nanoparticles (diameter <150 nm), the centrifugation–decantation step was not required as the interference was less than with larger particles. Parallel measurements with a ChemiDoc MP plate scanner gave indications of aggregation; however, the results were less sensitive than the absorbance measurements

Relating Redox Properties of Polyvinylamine‑<i>g</i>‑TEMPO/Laccase Hydrogel Complexes to Cellulose Oxidation

The structure and electrochemical properties of adsorbed complexes based on mixtures of polyvinylamine-<i>g</i>-TEMPO (PVAm-T) and laccase were related to the ability of the adsorbed complexes to oxidize cellulose. PVAm-T10 with 10% of the amines bearing TEMPO moieties (i.e., DS = 10%), adsorbed onto gold sulfonate EQCM-D sensor surfaces giving a hydrogel film that was 7 nm thick, 89% water, and encasing laccase (200 mM) and TEMPO moieties (33 mM). For DS values >10%, all of the TEMPOs in the hydrogel film were redox-active in that they could be oxidized by the electrode. With hydrogel layers made with lower-DS PVAm-Ts, only about half of the TEMPOs were redox-active; 10% DS appears to be a percolation threshold for complete TEMPO-to-TEMPO electron transport. In parallel experiments with hydrogel complexes adsorbed onto regenerated cellulose films, the aldehyde concentrations increased monotonically with the density of redox-active TEMPO moieties in the adsorbed hydrogel. The maximum density of aldehydes was 0.24 μmol/m², about 10 times less than the theoretical concentration of primary hydroxyl groups exposed on crystalline cellulose surfaces. Previous work showed that PVAm-T/laccase complexes are effective adhesives between wet cellulose surfaces when the DS is >10%. This work supports the explanation that TEMPO-to-TEMPO electron transport is required for the generation of aldehydes necessary for wet adhesion to PVAm

Sentinel Wraps: Real-Time Monitoring of Food Contamination by Printing DNAzyme Probes on Food Packaging

Here, we report the development of a transparent, durable, and flexible sensing surface that generates a fluorescence signal in the presence of a specific target bacterium. This material can be used in packaging, and it is capable of monitoring microbial contamination in various types of food products in real time without having to remove the sample or the sensor from the package. The sensor was fabricated by covalently attaching picoliter-sized microarrays of an E. coli-specific RNA-cleaving fluorogenic DNAzyme probe (RFD-EC1) to a thin, flexible, and transparent cyclo-olefin polymer (COP) film. Our experimental results demonstrate that the developed (RFD-EC1)-COP surface is specific, stable for at least 14 days under various pH conditions (pH 3–9), and can detect E. coli in meat and apple juice at concentrations as low as 10³ CFU/mL. Furthermore, we demonstrate that our sensor is capable of detecting bacteria while still attached to the food package, which eliminates the need to manipulate the sample. The developed biosensors are stable for at least the shelf life of perishable packaged food products and provide a packaging solution for real-time monitoring of pathogens. These sensors hold the potential to make a significant contribution to the ongoing efforts to mitigate the negative public-health-related impacts of food-borne illnesses

Facile Supermolecular Aptamer Inhibitors of L-Selectin

<div><p>Multivalent interactions occur frequently in nature, where they mediate high-affinity interactions between cells, proteins, or molecules. Here, we report on a method to generate multivalent aptamers (Multi-Aptamers) that target L-selectin function using rolling circle amplification (RCA). We find that the L-selectin Multi-Aptamers have increased affinity compared to the monovalent aptamer, are specific to L-selectin, and are capable of inhibiting interactions with endogenous ligands. In addition, the Multi-Aptamers efficiently inhibit L-selectin mediated dynamic adhesion in vitro and homing to secondary lymphoid tissues in vivo. Importantly, our method of generating multivalent materials using RCA avoids many of the challenges associated with current multivalent materials in that the Multi-Aptamers are high affinity, easily produced and modified, and biocompatible. We anticipate that the Multi-Aptamers can serve as a platform technology to modulate diverse cellular processes.</p></div

The Multi-Aptamer does not induce apoptosis or affect cell viability.

<p>(A) Jurkat cells were untreated or treated with SC-Multi-Aptamer, LS-Multi-Aptamer. Treatment with cyclosporine A (CA) served as a control. Apoptosis was assessed at the indicated time points by flow cytometry analysis of annexin V and propidium iodide (PI). (B) To assess potential effects on cell viability or proliferation, Jurkat cells were treated with the indicated compounds. Cell viability was assessed by introduction of XTT reagent. Treatment with 50 μM CA served as a control. Error bars are standard error of the mean (SEM).</p

LS-Multi-Aptamer inhibits dynamic adhesion and homing in vitro and in vivo.

<p>(A) Representative images of Jurkat cells after dynamic adhesion to activated endothelial cells and treatment with the indicated aptamers or Multi-Aptamers. Scale bar is 20 μm. (B) Quantification of Jurkat cells that dynamically adhered to activated endothelial cells following the indicated treatments. * p < 0.05; ** p < 0.01. (C) Quantification of relative recruitment of Jurkat cells to the mesenteric lymph nodes following treatment with the monovalent SC- or LS-aptamer or SC- or LS-Multi-Aptamer, normalized to the respective control. Error bars are SEM.</p

LS-Multi-Aptamer specifically binds to L-selectin on Jurkat cells.

<p>(A) Flow cytometry histograms of Jurkat cells stained with the indicated Multi-Aptamers and controls. (B) Confocal microscope images demonstrate that FITC-labeled LS-Multi-Aptamer (green) effectively binds to Jurkat cells (red). (C) Confocal microscope images demonstrate that cross-linking of multiple Jurkat cells by the LS-Multi-Aptamer can be occasionally found. Scale bar is 5 μm.</p

DNA sequences used in this study.

<p>DNA sequences used in this study.</p

LS-Multi-Aptamer has a higher binding affinity for L-selectin than the monovalent aptamer.

<p>(A) Jurkat cells were incubated with fluorescent monovalent L-selectin aptamer (LS-Aptamer) or the LS-Multi-Aptamer at the indicated concentrations and fluorescence assessed with flow cytometry. (B) Jurkat cells were simultaneously treated with FITC-labeled blocking antibody, DREG56 (100 nM) and increasing concentrations of the indicated reagents. The mean fluorescence for each sample is normalized to the mean fluorescence of the untreated sample labeled with FITC-DREG56.</p

Synthesis of the Multi-Aptamers.

<p>Following a 10 minute RCA reaction, the LS- and SC-Multi-Aptamer DNA products were only generated in the presence of the primer (LS, SC). The negative sample (-) does not contain a primer.</p