A novel cell-based biosensing platform (Large-scale Homogeneous Nanoelectrode Arryas, LHONA) is developed using a combination of sequential laser interference lithography and electrochemical deposition methods. This enables the sensitive discrimination of dopaminergic cells from other types of neural cells in a completely non-destructive manner owing to its enhanced biocompatibility and excellent electrochemical properties. As such, this platform/detection strategy holds great potential as an effective non-invasive in situ monitoring tool that can be used to determine stem cell fate for various regenerative applications.

Gold nanostars, functionalized with thiolated DNA hairpins bearing a Raman-active fluorescent dye at the 3′ terminus, were engineered to identify and quantify RNA mutations in the influenza A virus (IAV) genome employing surface enhanced Raman spectroscopy (SERS). The DNA hairpin structure was designed to selectively extend/fold in the absence/presence of the viral RNA targets, resulting in the fluorophore being brought away from or close to the gold nanostar surface, leading to an "OFF-ON" switching of the SERS signal. Validation of the switchable SERS nanostar probes was first carried out in buffer, showing that the detection is sequencespecific and that the high sensitivity provided by these SERS probes allows target detection at the single particle level. We also demonstrate that the degree of signal recovery can be closely correlated with the number of genetic mutations. Further experiments carried out with HeLa cell lysate spiked with RNA oligonucleotides demonstrate that the functionality of these nanoprobes were not detrimentally affected by the complex matrix. As a proof of concept, we also tested these nanoparticle probes in vitro by specifically targeting the hemagglutinin (HA) segment in live HeLa cells transfected with plasmids coding for either HA or two other IAV segments, PB1 and PB2, as negative controls. The intracellular SERS response in individual transfected HeLa cells demonstrates high sequenceselectivity of the probes for the HA segment, suggesting the applicability of these probes for multiplexed detection and quantification of viral RNAs in individual cells with an approach that can account for the viral population diversity. This also represents the first time that molecular beacon-based SERS probes have been employed to detect viral RNA target in intact individual cells.

Selective detection and precise quantification of biomolecules in intracellular settings play a pivotal role in the diagnostics and therapeutics of diseases, including various cancers and infectious epidemics. Because of this clinical relevance, nanoprobes with high sensitivity, wide tunability, and excellent biological stability have become of high demand. In particular, nanoflares based on gold nanoparticles have emerged as an attractive candidate for intracellular detection due to their efficient cellular uptake, enhanced binding affinity with complementary targets, and improved biological compatibility. However, nanoprobes, including these nanoflares, are known to be susceptible to the adsorption of proteins present in the biological environment, which leads to the formation of a so-called protein corona layer on their surface, leading to an altered targeting efficiency and cellular uptake. In this work, we leverage the nanoflares platform to demonstrate the effect of protein corona on biomolecular detection, quantification, as well as biological stability against enzymatic degradation. Nanoflares incubated in a biologically relevant concentration of serum albumin proteins (0.50 wt %) were shown to result in more than 20% signal reduction in target detection, with a decrease varying proportionally with the protein concentrations. In addition, similar signal reduction was observed for different serum proteins, and PEG backfilling was found to be ineffective in mitigating the negative impact induced by the corona formation. Furthermore, nuclease resistance in nanoflares was also severely compromised by the presence of the corona shell (∼2-fold increase in hydrolysis activity). This work demonstrates the consequences of an in situ formed protein corona layer on molecular detection/quantification and biological stability of nanoflares in the presence of nuclease enzymes, highlighting the importance of calibrating similar nanoprobes in proper biological media to improve the accuracy of molecular detection and quantification.

Even though gene repression is a powerful approach to exogenously regulate cellular behavior, developing a platform to effectively repress targeted genes, especially for stem cell applications, remains elusive. Herein, we introduce a nanomaterial-based platform that is capable of mimicking the function of transcription repressor proteins to downregulate gene expression at the transcriptional level for enhancing stem cell differentiation. We developed the NanoScript platform by integrating multiple gene repression molecules with a nanoparticle. First, we show a proof-of-concept demonstration using a GFP-specific NanoScript to knockdown GFP expression in neural stem cells (NSCs-GFP). Then, we show that a Sox9-specific NanoScript can repress Sox9 expression to initiate enhanced differentiation of NSCs into functional neurons. Overall, the tunable properties and gene knockdown capabilities of NanoScript enables its utilization for gene-repression applications in stem cell biology.

Even though gene repression is apowerfulapproach to exogenously regulate cellular behavior,d eveloping ap latform to effectively repress targeted genes,e specially for stemcell applications,remains elusive.Herein, we introduce ananomaterial-based platform that is capable of mimicking the function of transcription repressor proteins to downregulate gene expression at the transcriptional level for enhancing stemcell differentiation. We developed the "NanoScript" platform by integrating multiple gene repression molecules with ananoparticle.F irst, we show ap roof-of-concept demonstration using aG FP-specific NanoScript to knockdownG FP expression in neural stem cells (NSCs-GFP). Then, we showt hat aS ox9-specific NanoScript can repress Sox9 expression to initiate enhanced differentiation of NSCs into functional neurons.Overall, the tunable properties and gene-knockdown capabilities of NanoScript enables its utilization for generepression applications in stem cell biology.