Clinical validation of a novel automated cell-free DNA screening assay for trisomies 21, 13, and 18 in maternal plasma.

OBJECTIVE: To evaluate clinical performance of a new automated cell-free (cf)DNA assay in maternal plasma screening for trisomies 21, 18, and 13, and to determine fetal sex. METHOD: Maternal plasma samples from 1200 singleton pregnancies were analyzed with a new non-sequencing cfDNA method, which is based on imaging and counting specific chromosome targets. Reference outcomes were determined by either cytogenetic testing, of amniotic fluid or chorionic villi, or clinical examination of neonates. RESULTS: The samples examined included 158 fetal aneuploidies. Sensitivity was 100% (112/112) for trisomy 21, 89% (32/36) for trisomy 18, and 100% (10/10) for trisomy 13. The respective specificities were 100%, 99.5%, and 99.9%. There were five first pass failures (0.4%), all in unaffected pregnancies. Sex classification was performed on 979 of the samples and 99.6% (975/979) provided a concordant result. CONCLUSION: The new automated cfDNA assay has high sensitivity and specificity for trisomies 21, 18, and 13 and accurate classification of fetal sex, while maintaining a low failure rate. The study demonstrated that cfDNA testing can be simplified and automated to reduce cost and thereby enabling wider population-based screening.

Imaging single DNA molecules for high precision NIPT.

Cell-free DNA analysis is becoming adopted for first line aneuploidy screening, however for most healthcare programs, cost and workflow complexity is limiting adoption of the test. We report a novel cost effective method, the Vanadis NIPT assay, designed for high precision digitally-enabled measurement of chromosomal aneuploidies in maternal plasma. Reducing NIPT assay complexity is achieved by using novel molecular probe technology that specifically label target chromosomes combined with a new readout format using a nanofilter to enrich single molecules for imaging and counting without DNA amplification, microarrays or sequencing. The primary objective of this study was to assess the Vanadis NIPT assay with respect to analytical precision and clinical feasibility. Analysis of reference DNA samples indicate that samples which are challenging to analyze with low fetal-fraction can be readily detected with a limit of detection determined at <2% fetal-fraction. In total of 286 clinical samples were analysed and 30 out of 30 pregnancies affected by trisomy 21 were classified correctly. This method has the potential to make cost effective NIPT more widely available with more women benefiting from superior detection and false positive rates.

Nitric Oxide Dependent Degradation of Polyethylene Glycol-Modified Single-Walled Carbon Nanotubes: Implications for Intra-Articular Delivery.

Polyethylene glycol (PEG)-modified carbon nanotubes have been successfully employed for intra-articular delivery in mice without systemic or local toxicity. However, the fate of the delivery system itself remains to be understood. In this study 2 kDa PEG-modified single-walled carbon nanotubes (PNTs) are synthesized, and trafficking and degradation following intra-articular injection into the knee-joint of healthy mice are studied. Using confocal Raman microspectroscopy, PNTs can be imaged in the knee-joint and are found to either egress from the synovial cavity or undergo biodegradation over a period of 3 weeks. Raman analysis discloses that PNTs are oxidatively degraded mainly in the chondrocyte-rich cartilage and meniscus regions while PNTs can also be detected in the synovial membrane regions, where macrophages can be found. Furthermore, using murine chondrocyte (ATDC-5) and macrophage (RAW264.7) cell lines, biodegradation of PNTs in activated, nitric oxide (NO)-producing chondrocytes, which is blocked upon pharmacological inhibition of inducible nitric oxide synthase (iNOS), can be shown. Biodegradation of PNTs in macrophages is also noted, but after a longer period of incubation. Finally, cell-free degradation of PNTs upon incubation with the peroxynitrite-generating compound, SIN-1 is demonstrated. The present study paves the way for the use of PNTs as delivery systems in the treatment of diseases of the joint.

Graphene oxide is degraded by neutrophils and the degradation products are non-genotoxic.

Neutrophils were previously shown to digest oxidized carbon nanotubes through a myeloperoxidase (MPO)-dependent mechanism, and graphene oxide (GO) was found to undergo degradation when incubated with purified MPO, but there are no studies to date showing degradation of GO by neutrophils. Here we produced endotoxin-free GO by a modified Hummers' method and asked whether primary human neutrophils stimulated to produce neutrophil extracellular traps or activated to undergo degranulation are capable of digesting GO. Biodegradation was assessed using a range of techniques including Raman spectroscopy, transmission electron microscopy, atomic force microscopy, and mass spectrometry. GO sheets of differing lateral dimensions were effectively degraded by neutrophils. As the degradation products could have toxicological implications, we also evaluated the impact of degraded GO on the bronchial epithelial cell line BEAS-2B. MPO-degraded GO was found to be non-cytotoxic and did not elicit any DNA damage. Taken together, these studies have shown that neutrophils can digest GO and that the biodegraded GO is non-toxic for human lung cells.

Off-stoichiometry improves the photostructuring of thiol-enes through diffusion-induced monomer depletion.

Thiol-enes are a group of alternating copolymers with highly ordered networks and are used in a wide range of applications. Here, "click" chemistry photostructuring in off-stoichiometric thiol-enes is shown to induce microscale polymeric compositional gradients due to species diffusion between non-illuminated and illuminated regions, creating two narrow zones with distinct compositions on either side of the photomask feature boundary: a densely cross-linked zone in the illuminated region and a zone with an unpolymerized highly off-stoichiometric monomer composition in the non-illuminated region. Using confocal Raman microscopy, it is here explained how species diffusion causes such intricate compositional gradients in the polymer and how off-stoichiometry results in improved image transfer accuracy in thiol-ene photostructuring. Furthermore, increasing the functional group off-stoichiometry and decreasing the photomask feature size is shown to amplify the induced gradients, which potentially leads to a new methodology for microstructuring.

Biodegradation of single-walled carbon nanotubes by eosinophil peroxidase.

Eosinophil peroxidase (EPO) is one of the major oxidant-producing enzymes during inflammatory states in the human lung. The degradation of single-walled carbon nanotubes (SWCNTs) upon incubation with human EPO and H2O2 is reported. Biodegradation of SWCNTs is higher in the presence of NaBr, but neither EPO alone nor H2O2 alone caused the degradation of nanotubes. Molecular modeling reveals two binding sites for SWCNTs on EPO, one located at the proximal side (same side as the catalytic site) and the other on the distal side of EPO. The oxidized groups on SWCNTs in both cases are stabilized by electrostatic interactions with positively charged residues. Biodegradation of SWCNTs can also be executed in an ex vivo culture system using primary murine eosinophils stimulated to undergo degranulation. Biodegradation is proven by a range of methods including transmission electron microscopy, UV-visible-NIR spectroscopy, Raman spectroscopy, and confocal Raman imaging. Thus, human EPO (in vitro) and ex vivo activated eosinophils mediate biodegradation of SWCNTs: an observation that is relevant to pulmonary responses to these materials.

Hydrophobic pore array surfaces: wetting and interaction forces in water/ethanol mixtures.

Interactions between and wetting behavior of structured hydrophobic surfaces using different concentrations of water/ethanol mixtures have been investigated. Silica surfaces consisting of pore arrays with different pore spacings and pore depths were made hydrophobic by silanization. Their static and dynamic contact angles were found to be independent of the pore depth while fewer pores on the surface, i.e. a closer resemblance to a flat surface, gave a lower contact angle. As expected, a higher amount of ethanol facilitated wetting on all the surfaces tested. Confocal Raman microscopy measurements proved both water and ethanol to penetrate into the pores. AFM colloidal probe force measurements clearly showed that formation of air cavitation was hindered between the hydrophobic surfaces in presence of ethanol, and an increase in ethanol concentration was followed by a smaller jump-in distance and a weaker adhesion force. On separation, an immediate jump-out of contact occurred. The measured forces were interpreted as being due to capillary condensation of ethanol between the surfaces giving rise to very unstable cavities immediately rupturing on surface separation.

Effect of surface depressions on wetting and interactions between hydrophobic pore array surfaces.

The surface structure is known to significantly affect the long-range capillary forces between hydrophobic surfaces in aqueous solutions. It is, however, not clear how small depressions in the surface will affect the interaction. To clarify this, we have used the AFM colloidal probe technique to measure interactions between hydrophobic microstructured pore array surfaces and a hydrophobic colloidal probe. The pore array surfaces were designed to display two different pore spacings, 1.4 and 4.0 µm, each with four different pore depths ranging from 0.2 to 12.0 µm. Water contact angles measured on the pore array surfaces are lower than expected from the Cassie-Baxter and Wenzel models and not affected by the pore depth. This suggests that the position of the three-phase contact line, and not the interactions underneath the droplet, determines the contact angle. Confocal Raman microscopy was used to investigate whether water penetrates into the pores. This is of importance for capillary forces where both the movement of the three-phase contact line and the situation at the solid/liquid interface influence the stability of bridging cavities. By analyzing the shape of the force curves, we distinguish whether the cavity between the probe and the surfaces was formed on a flat part of the surface or in close proximity to a pore. The pore depth and pore spacing were both found to statistically influence the distance at which cavities form as surfaces approach each other and the distance at which cavities rupture during retraction.