Development of a rapid and ultra-sensitive RNA:DNA hybrid immunocapture based biosensor for visual detection of SARS-CoV-2 RNA.

The Development of reliable and field-compatible detection methods is essential to monitoring and controlling the spread of any global pandemic. We herein report a novel anti-RNA:DNA hybrid (anti-RDH) antibody-based biosensor for visual, colorimetric lateral flow assay, using gold nanoparticles, coupled with transcription-mediated-isothermal-RNA-amplification (TMIRA) for specific and sensitive detection of viral RNA. We have demonstrated its utility for SARS-CoV-2 RNA detection. This technique, which we have named RDH-LFA (anti-RNA:DNA hybrid antibody-based lateral flow assay), exploits anti-RDH antibody for immunocapture of viral RNA hybridized with specific DNA probes in lateral flow assay. This method uses biotinylated-oligonucleotides (DNAB) specific to SARS-CoV-2 RNA (vRNA) to generate a vRNA-DNAB hybrid. The biotin-tagged vRNA-DNAB hybrid molecules bind to streptavidin conjugated with gold nanoparticles. This hybrid complex is trapped by the anti-RDH antibody immobilized on the nitrocellulose membrane resulting in pink color signal leading to visual naked-eye detection in 1 minute. Combining RDH-LFA with isothermal RNA amplification (TMIRA) significantly improves the sensitivity (LOD:10 copies/µl) with a total turnaround time of an hour. More importantly, RDH-LFA coupled with the TMIRA method showed 96.6% sensitivity and 100% specificity for clinical samples when compared to a commercial gold standard reverse-transcription quantitative polymerase-chain-reaction assay. Thus, the present study reports a rapid, sensitive, specific, and simple method for visual detection of viral RNA, which can be used at the point-of-care without requiring sophisticated instrumentation.

A method for in situ visualization of Protein-Nascent RNA interactions in single cell using Proximity Ligation Assay (IPNR-PLA) in mammalian cells.

Proximity ligation assay (PLA) is an immunofluorescence assay, which determines in situ interaction of two biomolecules present within 40 nm close proximity. Here, we describe a modification of PLA for visual detection of in situ protein interactions with nascent RNA in a single cell (IPNR-PLA). In IPNR-PLA, nascent RNA is labeled by incorporating 5-fluorouridine (FU), a uridine nucleotide analogue, followed by covalent cross-linking of the interacting partners in proximity to newly synthesized RNA. By using combination of anti-BrdU antibody, which specifically binds to FU, and primary antibody against a protein of interest, the IPNR reaction results in fluorescent puncta as a positive signal, only if the candidate proteins are in proximity to nascent RNA. We have validated this method by demonstrating known CDK9 and elongating RNA pol II interaction with nascent RNA. Finally, we used this method to test for the presence of DNA double strand breaks as well as Poly (ADP-ribose) polymerase 1 (PARP1), an RNA binding protein, in the vicinity of nascent RNA in cancer cells. The capability of performing parallel IF labeling and quantifiable multiparameter measurements within heterogeneous cell populations makes IPNR-PLA very attractive for use in biological studies. Overall, we have developed the IPNR-PLA method for analysis of protein association with nascent RNA with single-cell resolution, which is highly sensitive, quantitative, efficient, and requires little starting experimental material.

Band gap and defect engineering of bismuth vanadate using La, Ce, Zr dopants to obtain a photoelectrochemical system for ultra-sensitive detection of glucose in blood serum.

Bismuth vanadate (BiVO4) is a promising photoactive material for the design of photoelectrochemical (PEC) analytical devices for the non-enzymatic detection of glucose. In this work, un-doped and La/Ce/Zr doped BiVO4 photo anodes were developed by spray pyrolysis coating to generate unique 2D hierarchical architectures using the facile ultrasonic spray coating technique without any complex pre or post-treatment. The influence of different dopants on the morphology and photoelectrochemical activity of BiVO4 coatings was investigated. X-ray diffraction, scanning electron microscopy, UV-vis optical absorbance, and positron annihilation techniques were used to evaluate the structure, defects, and optical properties of BiVO4 films. DFT simulation confirmed the Zr doping induced band gap reduction in the BiVO4 lattice. The Zr doping on the Bi site in BiVO4 lattice provided significantly low Bi and V-based defect density and a higher bulk diffusion length of charge pairs (4 times that of pristine) as well as charge transfer efficiency and this led to the foremost photocurrent for water splitting. The Zr-doped BiVO4 photo anode showed remarkable sensitivity in glucose sensing. The sensitivity and limit of detection of the Zr-doped BiVO4 PEC device towards glucose were 0.14 mA cm-2 mM-1 and 1.22 µM, respectively, in the concentration range of 1-7 mM. The system showed sensitive detection of glucose in blood serum. This is the first time that a 2D morphology electrode design consisting of Zr-doped BiVO4, which leads to exceptionally high sensitivity for glucose sensing, has been reported.

Emerging roles of bromodomain protein 4 in regulation of stem cell identity.

Understanding the mechanism of fate decision and lineage commitment is the key step for developing novel stem cell applications in therapeutics. This process is coordinately regulated through systematic epigenetic reprogramming and concomitant changes in the transcriptional landscape of the stem cells. One of the bromo- and extra-terminal domain (BET) family member proteins, bromodomain protein 4 (BRD4), performs the role of epigenetic reader and modulates gene expression by recruiting other transcription factors and directly regulating RNA polymerase II elongation. Controlled gene regulation is the critical step in maintenance of stem cell potency and dysregulation may lead to tumor formation. As a key transcriptional factor and epigenetic regulator, BRD4 contributes to stem cell maintenance in several ways. Being a druggable target, BRD4 is an attractive candidate for exploiting its potential in stem cell therapeutics. Therefore, it is crucial to elucidate how BRD4, through its interplay with pluripotency transcriptional regulators, control lineage commitment in stem cells. Here, we systemically review the role of BRD4 in complex gene regulatory network during three specific states of stem cell transitions: cell differentiation, cell reprogramming and transdifferentiation. A thorough understanding of BRD4 mediated epigenetic regulation in the maintenance of stem cell potency will be helpful to strategically control stem cell fates in regenerative medicine.

Label-free rapid electrochemical detection of DNA hybridization using ultrasensitive standalone CNT aerogel biosensor.

We report the development of an ultrasensitive label-free DNA biosensor device with fully integrated standalone carbon nanotube (CNT) aerogel electrode. The multi-directional tenuous network of clustered CNT embedding into the CNT aerogel electrode demonstrates linear ohmic and near isotropic electrical properties, thereby providing high sensitivity for nucleic acid detection. Using this device, the target DNA hybridization is detected by a quantifiable change in the electrochemical impedance, with a distinct response to the single-stranded probe alone or double-stranded target-probe complex. The target DNA is specifically detected with limit of detection (LoD) of 1 pM with a turnaround time of less than 20 min, which is unprecedented for a miniaturized CNT aerogel sensor and impedance spectroscopy without an intermediate DNA amplification step. Moreover, this system is able to differentiate between the closely related target sequences by the distinct impedance response rendering it highly specific. To the best of our knowledge, this is the first report showing the use of standalone bare CNT aerogel electrode without any substrate support, coupled with electrochemical impedance spectroscopy, for the detection of DNA hybridization. Altogether, the results show that our system is fast, sensitive and specific for label-free rapid direct DNA detection, promising a novel avenue for bio-sensing.

Spontaneous Formation of Cationic Vesicles in Aqueous DDAB-Lecithin Mixtures for Efficient Plasmid DNA Complexation and Gene Transfection.

Cationic liposomes have become an attractive tool to deliver genes and interfering RNA into cells. Herein, we report the application of spontaneously formed cationic vesicles in mixtures of lecithin and cationic amphiphiles for efficient transfection of plasmid DNA and siRNA into cells. The average hydrodynamic diameter of the phospholipid vesicles was modulated by changing the ratio of dihexadecyldimethylammonium bromide (DDAB) to phospholipid in the vesicles. The vesicles were characterized by dynamic light scattering, ζ potential, and small-angle X-ray scattering. Depending on the ratio of DDAB to phospholipid, the average size of the vesicles can be varied in the range of 150-300 nm with a ζ potential of +40 mV. The ability of these cationic vesicles to form lipoplexes upon binding with pDNA is demonstrated by ζ potential, isothermal titration calorimetry, gel retardation, and DNase I digestion assay. The enthalpy of binding between pDNA and cationic liposome was found to be -5.7 (±0.8) kJ/mol. The cellular uptake studies of lipoplexes observed by fluorescence microscopy confirmed good transfection efficiency of DDAB liposomes in MCF-7 and HeLa cells. The fluorescent imaging analysis showed effective gene delivery and expression of green fluorescent protein. In addition, the formulation has demonstrated an ability to deliver small interfering RNA (siBRD4) for efficient gene silencing as seen by a significant decrease in BRD4 protein level in siBRD4-treated cells. Comparison of the transfection efficiency of different formulations suggests that DDAB-rich mixed phospholipid vesicles with size <200 nm are better than large size vesicles for improved endocytosis and gene expression.