Sub-Nanomolar Detection of Oligonucleotides Using Molecular Beacons Immobilized on Lightguiding Nanowires.

The detection of oligonucleotides is a central step in many biomedical investigations. The most commonly used methods for detecting oligonucleotides often require concentration and amplification before detection. Therefore, developing detection methods with a direct read-out would be beneficial. Although commonly used for the detection of amplified oligonucleotides, fluorescent molecular beacons have been proposed for such direct detection. However, the reported limits of detection using molecular beacons are relatively high, ranging from 100 nM to a few µM, primarily limited by the beacon fluorescence background. In this study, we enhanced the relative signal contrast between hybridized and non-hybridized states of the beacons by immobilizing them on lightguiding nanowires. Upon hybridization to a complementary oligonucleotide, the fluorescence from the surface-bound beacon becomes coupled in the lightguiding nanowire core and is re-emitted at the nanowire tip in a narrower cone of light compared with the standard 4π emission. Prior knowledge of the nanowire positions allows for the continuous monitoring of fluorescence signals from each nanowire, which effectively facilitates the discrimination of signals arising from hybridization events against background signals. This resulted in improved signal-to-background and signal-to-noise ratios, which allowed for the direct detection of oligonucleotides at a concentration as low as 0.1 nM.

Enhanced human T cell expansion with inverse opal hydrogels.

Advanced personalized immunotherapies still have to overcome several biomedical and technical limitations before they become a routine cancer treatment in spite of recent achievements. In adoptive cell therapy (ACT), the capacity to obtain adequate numbers of therapeutic T cells in the patients following ex vivo treatment should be improved. Moreover, the time and costs to produce these T cells should be reduced. In this work, inverse opal (IOPAL) 3D hydrogels consisting of poly(ethylene) glycol (PEG) covalently combined with heparin were engineered to resemble the environment of lymph nodes, where T cells get activated and proliferate. The introduction of an IOPAL strategy allowed a precise control on the porosity of the hydrogels, providing an increase in the proliferation of primary human CD4+ T cells, when compared with state-of-the-art expansion systems. Additionally, the IOPAL hydrogels also showed a superior expansion compared to hydrogels with the same composition, but without the predetermined pore structure. In summary, we have shown the beneficial effect of having an IOPAL architecture in our 3D hydrogels to help achieving large numbers of cells, while maintaining the desired selected phenotypes required for ACT.

Enhanced Optical Biosensing by Aerotaxy Ga(As)P Nanowire Platforms Suitable for Scalable Production.

Sensitive detection of low-abundance biomolecules is central for diagnostic applications. Semiconductor nanowires can be designed to enhance the fluorescence signal from surface-bound molecules, prospectively improving the limit of optical detection. However, to achieve the desired control of physical dimensions and material properties, one currently uses relatively expensive substrates and slow epitaxy techniques. An alternative approach is aerotaxy, a high-throughput and substrate-free production technique for high-quality semiconductor nanowires. Here, we compare the optical sensing performance of custom-grown aerotaxy-produced Ga(As)P nanowires vertically aligned on a polymer substrate to GaP nanowires batch-produced by epitaxy on GaP substrates. We find that signal enhancement by individual aerotaxy nanowires is comparable to that from epitaxy nanowires and present evidence of single-molecule detection. Platforms based on both types of nanowires show substantially higher normalized-to-blank signal intensity than planar glass surfaces, with the epitaxy platforms performing somewhat better, owing to a higher density of nanowires. With further optimization, aerotaxy nanowires thus offer a pathway to scalable, low-cost production of highly sensitive nanowire-based platforms for optical biosensing applications.