Rapid design and implementation of an adaptive pooling workflow for SARS-CoV-2 testing in an NHS diagnostic laboratory: a proof-of-concept study.

Background: Diagnostic laboratories are currently required to provide routine testing of asymptomatic staff and patients as a part of their clinical screening for SARS-CoV-2 infection. However, these cohorts display very different disease prevalence from symptomatic individuals and testing capacity for asymptomatic screening is often limited. Group testing is frequently proposed as a possible solution to address this; however, proposals neglect the technical and operational feasibility of implementation in a front-line diagnostic laboratory. Methods: Between October and December 2020, as a seven-week proof of concept, we took into account scientific, technical and operational feasibility to design and implement an adaptive pooling strategy in an NHS diagnostic laboratory in London (UK). We assessed the impact of pooling on analytical sensitivity and modelled the impact of prevalence on pooling strategy. We then considered the operational constraints to model the potential gains in capacity and the requirements for additional staff and infrastructure. Finally, we developed a LIMS-agnostic laboratory automation workflow and software solution and tested the technical feasibility of our adaptive pooling workflow. Results: First, we determined the analytical sensitivity of the implemented SARS-CoV-2 assay to be 250 copies/mL. We then determined that, in a setting with limited analyser capacity, the testing capacity could be increased by two-fold with pooling, however, in a setting with limited reagents, this could rise to a five-fold increase. These capacity increases could be realized with modest additional resource and staffing requirements whilst utilizing up to 76% fewer plastic consumables and 90% fewer reagents. Finally, we successfully implemented a plate-based pooling workflow and tested 920 patient samples using the reagents that would usually be required to process just 222 samples. Conclusions: Adaptive pooled testing is a scientifically, technically and operationally feasible solution to increase testing capacity in frontline NHS diagnostic laboratories.

Preparation and characterization of a pH- and thermally responsive poly(N-isopropylacrylamide-co-acrylic acid)/porous SiO(2) hybrid.

A multifunctional nanohybrid composed of a pH- and thermoresponsive hydrogel, poly(N-isopropylacrylamide-co-acrylic acid), poly(NIPAM-co-AAc) is synthesized in-situ within the mesopores of an oxidized porous Si template. The hybrid is characterized by electron microscopy and by thin film optical interference spectroscopy. The optical reflectivity spectrum of the hybrid displays Fabry-Pérot fringes characteristic of thin film optical interference, enabling direct, real-time observation of the pH- induced swelling and volume phase transitions associated with the confined poly(NIPAM-co-AAc) hydrogel. The optical response correlates to the percentage of AAc contained within the hydrogel, with a maximum change observed for samples containing 20% AAc. The swelling kinetics of the hydrogel are significantly altered due to the nanoscale confinement; displaying a more rapid response to pH or heating stimuli relative to bulk polymer films. The inclusion of AAc dramatically alters the thermoresponsiveness of the hybrid at pH 7, effectively eliminating the lower critical solution temperature (LCST). The observed changes in the optical reflectivity spectrum are interpreted in terms of changes in the dielectric composition and morphology of the hybrids.

Novel biodegradable lipid nano complex for siRNA delivery significantly improving the chemosensitivity of human colon cancer stem cells to paclitaxel.

BACKGROUND: Targeting of a specific subset of cells is mandatory for the successful application of siRNA mediated silencing in anticancer therapy. A recent theory suggests that colon cancer is sustained by a small subpopulation of cells, termed cancer stem cells (CSCs). These cells are characterized by their innate drug resistance properties, which is one of the key factors of chemotherapy failure. The goal of this study was to assess whether a novel siRNA delivery carrier, with an appropriate siRNA, targeted to CD133+ cells has the potential to improve the efficacy of conventional chemotherapy. METHODS: In this study, a novel synthetic siRNA carrier platform was designed and synthesized. This carrier was composed of a cationic oligomer (PEI(1200)), a hydrophilic polymer (polyethylene glycol) and a biodegradable lipid-based crosslinking moiety. Libraries of polymers were synthesized by varying their lipid composition. Their transfection efficacy was evaluated in vitro using CHOK1 cells. The polymer was characterized using molecular weight, particle encapsulation assay, particle size and surface charge analysis. RESULTS: It was demonstrated that the lipid composition in the polymer plays a critical role in transfection. Optimizing the physicochemical properties of the polymers is crucial in achieving favorable knockdown. Lipid nano complex with composition PEI-Lipid(1:16) was the optimum ratio for gene silencing. Additionally, silencing of multidrug resistance gene (MDR1) and treatment with paclitaxel play a synergistic role in increasing the efficacy as compared to the drug alone. CONCLUSIONS: In the present study a novel siRNA delivery carrier system with an MDR1-targeting siRNA (siMDR1) effectively reduced the expression of MDR1 in human colon CSCs (CD133+ enriched cell population), resulting in significantly increasing the chemosensitivity to paclitaxel.

pH-triggered release of vancomycin from protein-capped porous silicon films.

OBJECTIVE: An in vitro model system for pH-triggered release of the antibiotic vancomycin from porous Si films is studied. METHOD: Vancomycin is infused into a mesoporous Si film from a mixed aqueous/acetonitrile solution and trapped by a capping layer containing the protein bovine serum albumin (BSA). The protein effectively traps vancomycin in the porous nanostructure at pH 4.0; the protein dissolves and vancomycin is released into solution when the pH increases to 7.4. The surface chemistry of porous Si exerts a substantial effect on the efficacy of drug loading. The amount of drug loading is larger in freshly-etched (hydrophobic, hydrogen-terminated) porous Si and smaller in methyl-modified, undecylenic acid-modified and thermally oxidized samples. The quantity of drug loaded in a freshly etched porous Si chip is proportional to the thickness of the porous layer, which exhibits a constant volume loading efficiency of 31% (v/v). Flow-cell experiments designed to mimic the transition from pH 4 to 7 that occurs when material moves from the stomach to the upper intestinal tract were performed on the freshly etched films and vancomycin- and BSA-release rates were quantified from the effluent of the flow cell by high-pressure liquid chromatography analysis. RESULTS & CONCLUSION: There is a small, constant rate of vancomycin release at pH 4 that is independent of the amount of drug loaded in the pores. This is attributed to diffusion of vancomycin from the BSA-capping layer. The release rate increases five- to tenfold when the pH of the solution in the flow cell increases to 7.4; 100% of the drug is released within 3 h of this increase.

A simplified biomolecule attachment strategy for biosensing using a porous Si oxide interferometer.

A simple strategy for linking biomolecules to porous Si surfaces and detecting peptide/drug binding is described. Porous Si is prepared using an electrochemical etch and then thermally oxidized by heating in ambient atmosphere. Bovine serum albumin (BSA) is then non-covalently adsorbed to the inner pore walls of the porous Si oxide (PSiO(2)) matrix. The BSA layer is used as a linker for covalent attachment of the peptide Ac-L-Lysine-D-Alanine-D-Alanine (KAA) using published bioconjugation chemistry. BSA-coated surfaces functionalized with KAA display specificity for the glycopeptide vancomycin while resisting adsorption of non-specific reagents. While the biomolecule attachment strategy reported here is used to bind peptides, the scheme can be generalized to the linking of any primary amine-containing molecule to PSiO(2) surfaces.

Engineering the chemistry and nanostructure of porous silicon Fabry-Pérot films for loading and release of a steroid.

A method for engineering the surface chemistry and pore dimensions in porous Si films for the purpose of controlling the loading and release of a hydrophobic drug is described. Loading of the steroid dexamethasone is confirmed by Fourier transform infrared spectroscopy, and the release rates are characterized by observation of the appearance of the drug in solution (UV-vis absorption spectroscopy) and by measurement of the Fabry-Perot fringes in the optical reflectivity spectrum of the porous Si film. Optical reflectivity changes provide a measure of the release rate of the drug that is amenable to in-vivo diagnostic applications. Fresh porous Si films are prepared by electrochemical etch and subsequently modified by hydrosilylation with 1-dodecene. The dodecene-modified samples are more robust in aqueous environments and exhibit slower release rates of the drug relative to freshly etched porous Si. Whereas the relatively large dexamethasone molecule is found to infiltrate the freshly etched samples, it does not enter the chemically modified films, because of steric crowding from the dodecyl species. To achieve a high degree of loading into these modified films, the pores are enlarged before hydrosilylation by treatment with an aqueous solution containing HF and dimethyl sulfoxide. The pore expanded, chemically modified samples admit approximately 70% of the dexamethasone that can be admitted into an unmodified (freshly etched) sample. Diffusion of the steroid from the modified, pore expanded films into phosphate-buffered saline solution is slower than from the unmodified sample by a factor of approximately 20, with 90% of the drug delivered in 3 days for the chemically modified films compared to 3 h for the unmodified films.