Design and synthesis of novel palladium cyclometallate-based fluorescent probe: Studies on interaction with cell membrane by confocal and fluorescence lifetime imaging.

Coordination complexes offer great potential as cellular imaging probes, which allow to examine specific cell organelle structures in their physiological conditions to better understand the biological system. Understanding the heterogeneous nature of the cell membrane could unveil details of their functionality. Here, we have developed a new anthracene conjugated fluorescent palladium(II) cyclometallate [PdL1Cl] where L1H = [2-(2- (anthracen-9-ylmethylene)-1-phenylhydrazineyl)pyridine] (H stands for dissociable proton), which not only specifically stains the cell membrane, but could be utilized to visualise the membrane by the confocal and fluorescence lifetime imaging microscopy (FLIM). This probe is unable to enter inside the cell as it did not pass through the cell membrane via diffusion or various organic and metal transporters. However, the great lipophilicity of fluorescein improves the interaction of the probe with the peptidoglycan layer of the cell membrane. Probable dissociation of chloride ion and formation of positively charged palladium complex resulted in staining the negatively charged cell membrane. The 3D confocal imaging clearly expressed sole membrane staining by the probe. The probe efficiently stains both cancer cells (HeLa and MCF-7 cell lines) and normal cell (HEK 293 T), confirming the universality of the probe in membrane staining.

Super-Resolution Microscopy Revealed the Lysosomal Expansion During Epigallocatechin Gallate-Mediated Apoptosis.

Direct visualization of the dynamic events in lysosomes during drug-mediated programmed cell death (apoptosis) is a great challenge. This is due to the lack of resolving power of a conventional microscope and also the unavailability of a suitable multimodal probe that simultaneously can carry the drug with high loading capacity and ensure its specific internalization into lysosomes. In this work, using super-resolution microscopy, we observed the lysosomal expansion during apoptosis that was treated with epigallocatechin gallate (EGCG) conjugated to bovine serum albumin (BSA). Albumin protein is known to internalize into lysosomes via endocytosis, thus helping in the specific delivery of EGCG to the lysosomal compartment. The conjugation of EGCG to BSA not only helped in increasing the killing efficiency of cancer cells but it also reduces the side effects and produces minimal reactive oxygen species. The decrease in local viscosity helped in lysosomal expansion during apoptosis.

Absorption and emission of light in red emissive carbon nanodots.

The structure-function relationship, especially the origin of absorption and emission of light in carbon nanodots (CNDs), has baffled scientists. The multilevel complexity arises due to the large number of by-products synthesized during the bottom-up approach. By performing systematic purification and characterization, we reveal the presence of a molecular fluorophore, quinoxalino[2,3-b]phenazine-2,3-diamine (QXPDA), in a large amount (∼80% of the total mass) in red emissive CNDs synthesized from o-phenylenediamine (OPDA), which is one of the well-known precursor molecules used for CND synthesis. The recorded NMR and mass spectra tentatively confirm the structure of QXPDA. The close resemblance of the experimental vibronic progression and the mirror symmetry of the absorption and emission spectra with the theoretically simulated spectra confirm an extended conjugated structure of QXPDA. Interestingly, QXPDA dictates the complete emission characteristics of the CNDs; in particular, it showed a striking similarity of its excitation independent emission spectra with that of the original synthesized red emissive CND solution. On the other hand, the CND like structure with a typical size of ∼4 nm was observed under a transmission electron microscope for a blue emissive species, which showed both excitation dependent and independent emission spectra. Interestingly, Raman spectroscopic data showed the similarity between QXPDA and the dot structure thus suggesting the formation of the QXPDA aggregated core structure in CNDs. We further demonstrated the parallelism in trends of absorption and emission of light from a few other red emissive CNDs, which were synthesized using different experimental conditions.

Effect of Protein Corona on the Drug Delivery of Carbogenic Nanodots and Their Mapping by Fluorescence Lifetime Imaging Microscopy.

It is practically impossible to avoid the nonspecific binding of protein to a nanocarrier when it enters a biological fluid. This hinders the chemotherapeutic efficacy of the nanocarrier to a large extent. Surface functionalization, in the recent past, helped in reducing such nonspecific interactions. However, there is a lack of understanding as to how they help in the case of nanocarriers with size <6 nm. Here, we show that the glutathione and folic acid functionalization to a small carbogenic nanocarrier leads to substantial improvement in cell internalization and chemotherapeutic efficacy. The functionalization on smaller size of the nanocarrier helped in manipulating the binding affinity of the protein, which in turn helped in easy dynamic exchange with the surrounding environment. Using fluorescence lifetime imaging, we directly visualized and mapped the released drug at a very high resolution and provide a comprehensive mechanism of the drug distribution inside a cancer cell, as a consequence of the different affinity of protein corona on the carbon nanoparticle.

Fluorescent Probes for Super-Resolution Microscopy of Lysosomes.

Lysosomes are membrane-enclosed small spherical cytoplasmic organelles. Malfunctioning and abnormalities in lysosomes can cause a plethora of neurodegenerative diseases. Consequently, understanding the structural information on lysosomes down to a subnanometer level is essential. Recently, super-resolution imaging techniques enable us to visualize dynamical processes occurring in suborganelle structures inside living cells down to subnanometer accuracy by breaking the diffraction limit. A brighter and highly photostable fluorescent probe is essential for super-resolution microscopy. In this regard, this mini-review deals with the various types of super-resolution techniques and the probes that are used to specifically stain and resolve the structure of the lysosomes.

Bovine Serum Albumin-Conjugated Red Emissive Gold Nanocluster as a Fluorescent Nanoprobe for Super-resolution Microscopy.

The gold nanocluster (GNC), because of its interesting photoluminescence properties and easy renal clearance from the body, has tremendous biomedical applications. Unfortunately, it has never been explored for super-resolution microscopy (SRM). Here, we present a protein-conjugated red emissive GNC for super-resolution radial fluctuation (SRRF) of the lysosome in HeLa cells. The diameter of the lysosome obtained in SRRF is ∼59 nm, which is very close to the original diameter of the smallest lysosome in HeLa cells. Conjugation of protein to GNC aided in the specific labeling of the lysosome. We hope that GNC not only will replace some of the common dyes used in SRM but due to its electron beam contrast could also be used as a multimodal probe for several other correlative bioimaging techniques.

Serum albumin-mediated strategy for the effective targeting of SARS-CoV-2.

Novel coronavirus (NCoV-19), also known as SARS CoV-2, is a pathogen causing an emerging infection that rapidly increases in incidence and geographic range, is associated with the ever-increasing morbidity and mortality rates, and shows sever economic impact worldwide. The WHO declares the NCoV-19 infection disease (COVID-19) a Public Health Emergency of International Concern on 30 January 2020 and subsequently, on March 11, 2020, declared it a Global Pandemic. Although some people infected with SARS CoV-2 have no symptoms, the spectrum of symptomatic infection ranges from mild to critical, with most COVID-19 infections being not severe. The common mild symptoms include body aches, dry cough, fatigue, low-grade fever, nasal congestion, and sore throat. More severe COVID-19 symptoms are typical of pneumonia, and upon progression, the patient's condition can worsen with severe respiratory and cardiac problems. Currently, there is no drug or vaccine for curing patients. It has been observed that people with challenged immunity are highly prone to SARS CoV-2 infection and least likely to recover. Also, older adults and people of any age with serious underlying medical conditions might be at higher risk for severe forms of COVID-19. We are suggesting here a strategy for the COVID-19 treatment that could be effective in curing the patients in the current scenario when no efficient medicine or Vaccine is currently available, and Clinicians solely depend upon the performing trials with drugs with known antiviral activities. Our proposed strategy is based on the compilation of published scientific research and concepts. The different published research indicates the success of a similar strategy in different physiological conditions, and such a strategy is widely studied at the cellular level and in animal models.

Dual responsive specifically labelled carbogenic fluorescent nanodots for super resolution and electron microscopy.

Due to their high biocompatibility and nontoxic nature, carbogenic fluorescent nanodots (FNDs) have already shown their application in bioimaging. However, their non-specific labeling has restricted their application in live cell super resolution microscopy (SRM). Here we introduce, for the first time, an orange emissive FND, specifically conjugated to the HeLa cell actin filament, for successful single molecule stochastic optical reconstruction microscopy (STORM) and super resolution radial fluctuation (SRRF) microscopy. The resolution obtained in SRRF (∼35 nm) was almost an order of magnitude less than the diffraction limited spot. Interestingly, in addition, the FND also showed electron microscope (EM) contrast inside the cell. We hope that this FND will not only replace some of the common dyes used for SRM, but will also be used as a dual responsive marker in correlative super resolution microscopy (CLEM).

Carbon coated core-shell multifunctional fluorescent SPIONs.

Due to their unique magnetic properties, multiple surface functionality and biocompatibility, superparamagnetic iron oxide nanoparticles (SPIONs) show very promising characteristics as magnetic resonance (MR) contrast agents in biomedical applications. However, a lack of fluorescence makes SPIONs inappropriate for multimodal bioimaging. SPIONs surface functionalized by either organic fluorescent molecules or semiconductor quantum dots (QDs) have been reported as bioimaging probes but subsequent deterioration of the fluorescent dyes due to low photostability and quick photobleaching limits their long term practical application. In addition, QDs are found to be toxic in nature. Here, we present a novel one step method to synthesize non-toxic carbon coated highly photostable core-shell magnetic and fluorescent SPIONs with long-lasting fluorescence alongside a superior magnetic resonance (MR) imaging ability. Apart from the highly comparable superparamagnetic properties of the SPIONs, the optical response of the material is much better than commonly used Rhodamine or cyanine dyes.

Small molecular organic nanocrystals resemble carbon nanodots in terms of their properties.

The most commonly observed phenomena in carbon nanodots (CNDs) are the strong excitation wavelength dependent multicolor fluorescence emission and the particle size distribution between 3-5 nm observed using a transmission electron microscope (TEM). However, it is not evident yet whether the emission originates from the particles observed using a TEM. In this article, we show that hydrothermal treatment of citric acid produces methylenesuccinic acid, which gives rise to hydrogen-bonded nano-assemblies with CND-like properties. While single crystal X-ray crystallography confirms the structure of methylenesuccinic acid, fluorescence correlation spectroscopy (FCS) confirms the presence of a molecular fluorophore with an average hydrodynamic diameter of ∼0.9 nm. This size is much smaller than the size of the particles observed using a TEM. We conclude that the particles observed using a TEM are the drying mediated nanocrystals of methylenesuccinic acid.

Phase engineering of seamless heterophase homojunctions with co-existing 3R and 2H phases in WS2 monolayers.

Self-organized semiconductor-semiconductor heterostructures (3R-2H) that coexist in atomically thin 2D monolayers forming homojunctions are of great importance for next-generation nanoelectronics and optoelectronics applications. Herein, we investigated the defect controlled growth of heterogeneous electronic structure within a single domain of monolayer WS2 to enable in-plane homojunctions consisting of alternate 2H semiconducting and 3R semiconducting phases of WS2. X-ray photoelectron, Raman, and photoluminescence spectroscopy along with fluorescence and Kelvin probe force microscopy imaging confirm the formation of homojunctions, enabling a direct correlation between chemical heterogeneity and electronic heterostructure in the atomically thin WS2 monolayer. Quantitative analysis of phase fractions shows 59% stable 2H phase and 41% metastable 3R phase estimated over WS2 flakes of different sizes. Time-resolved fluorescence lifetime imaging confirms distinct contrast between 2H and 3R phases with two distinct lifetimes of 3.2 ns and 1.1 ns, respectively. Kelvin probe force microscopy imaging revealed an abrupt change in the contact potential difference with a depletion width of ∼2.5 µm, capturing a difference in work function of ∼40 meV across the homojunction. Further, the thermal stability of coexisting phases and their temperature dependent optical behavior show a distinct difference among 2H and 3R phases. The investigated aspects of the controlled in plane growth of coexisting phases with seamless homojunctions, their properties, and their thermal stability will enable the development of nanoscale devices that are free from issues of lattice mismatch and grain boundaries.

Charge-Driven Fluorescence Blinking in Carbon Nanodots.

This study focuses on the mechanism of fluorescence blinking of single carbon nanodots, which is one of their key but less understood properties. The results of our single-particle fluorescence study show that the mechanism of carbon nanodots blinking has remarkable similarities with that of semiconductor quantum dots. In particular, the temporal behavior of carbon nanodot blinking follows a power law both at room and at cryogenic temperatures. Our experimental data suggest that static quenching via Dexter-type electron transfer between surface groups of a nanoparticle plays a major role in the transition of carbon nanodots to off or gray states, whereas the transition back to on states is governed by an electron tunneling from the particle's core. These findings advance our understanding of the complex mechanism of carbon nanodots emission, which is one of the key steps for their application in fluorescence imaging.

Single-molecule analysis of fluorescent carbon dots towards localization-based super-resolution microscopy.

The advancement of high-resolution bioimaging has always been dependent on the discovery of bright and easily available fluorescent probes. Fluorescent carbon nanodots, an interesting class of relatively new nanomaterials, have emerged as a versatile alternative due to their superior optical properties, non-toxicity, cell penetrability and easy routes to synthesis. Although a plethora of reports is available on bioimaging using carbon dots, single-molecule-based super-resolution imaging is rare in the literature. In this study, we have systematically characterized the single-molecule fluorescence of three carbon dots and compared them with a standard fluorescent probe. Each of these carbon dots showed a long-lived dark state in the presence of an electron acceptor. The electron transfer mechanism was investigated in single-molecule as well as in ensemble experiments. The average on-off rate between the fluorescent bright and dark states, which is one of the important parameters for single-molecule localization-based super-resolution microscopy, was measured by changing the laser power. We report that the photon budget and on-off rate of these carbon dots were good enough to achieve single-molecule localization with a precision of ~35 nm.

Time-Resolved Emission Reveals Ensemble of Emissive States as the Origin of Multicolor Fluorescence in Carbon Dots.

The origin of photoluminescence in carbon dots has baffled scientists since its discovery. We show that the photoluminescence spectra of carbon dots are inhomogeneously broadened due to the slower relaxation of the solvent molecules around it. This gives rise to excitation-dependent fluorescence that violates the Kasha-Vavilov rule. The time-resolved experiment shows significant energy redistribution, relaxation among the emitting states, and spectral migration of fluorescence spectra in the nanosecond time scale. The excitation-dependent multicolor emission in time-integrated spectra is typically governed by the relative population of these emitting states.

Direct visualization of lead corona and its nanomolar colorimetric detection using anisotropic gold nanoparticles.

The study presents dithiothreitol (DTT) functionalized anisotropic gold nanoparticles (GNP) based colorimetric sensor for detection of toxic lead ions in water. Our results demonstrate the selectivity and sensitivity of the developed sensor over various heavy metal ions with detection limit of ∼9 nM. The mechanism of sensing is explained on the basis of unique corona formation around the DTT functionalized anisotropic GNP.

Orientational switching of protein conformation as a function of nanoparticle curvature and their geometrical fitting.

Among the various surface properties, nanoparticle curvature has a direct effect on the inner root of protein nanoparticle interaction. However, the orientation of adsorbed proteins onto the nanoparticle surface and its binding mechanism still remains elusive because of the lack of in-depth knowledge at the molecular level. Here, we demonstrate detail molecular insights of the orientational switching of several serum proteins as a function of nanoparticle curvature using theoretical simulation along with some experimental results. With the variation of binding stability, four distinctly different classes of orientation were observed for human serum albumin, whereas only two unique classes of conformations were observed for ubiquitin, insulin, and haemoglobin. As a general observation, our data suggested that orientations were exclusively dependent on the specific protein structure and the geometrical fitting onto the nanoparticle surface.

Functional molecular lumino-materials to probe serum albumins: solid phase selective staining through noncovalent fluorescent labeling.

Selective staining of human serum albumin protein in gel electrophoresis over wide range of other protein(s) is extremely important because it contains more than 60% volume of serum fluid in human body. Given the nonexistence of suitable dye materials for selective staining of serum albumins in gel electrophoresis, we report a new class of easy synthesizable and low molecular weight staining agents based on 3-amino-N-alkyl-carbazole scaffold for selective staining of serum albumins in solid phase. A detailed structure-efficiency relationship (SER) study enabled us to develop two such potent functional molecular probes which stain both human and bovine serum albumin selectively in gel electrophoresis in the presence of other proteins and enzymes. The present gel staining process was found to be very simple and less time-consuming as compared to the conventional coomassie blue staining which in turn makes these probes a new class of serum albumin-specific staining materials in proteome research. Moreover, these molecular lumino-materials can detect serum albumins at subnanomolar level in the presence of broad spectrum of other proteins/enzymes in aqueous buffer (99.9% water, pH = 7.3) keeping the protein secondary structure intact. Our experimental and the docking simulation results show that these probes bind preferentially at 'binding site I' of both the serum proteins.

Binding of hairpin polyamides to DNA studied by fluorescence correlation spectroscopy for DNA nanoarchitectures.

We have recently constructed a "DNA strut" consisting of two DNA-binding hairpin polyamides of Dervan-type connected via a long flexible linker and were able to show that this strut can be used to sequence-selectively connect DNA helices. This approach provides a second structural element (besides the Watson-Crick base pairing) for the assembly of higher-order DNA nanoarchitectures from smaller DNA building blocks. Since none of the existing analytical techniques for studying this kind of system were found suitable for detection and quantification of the formation of the resulting complexes, we chose fluorescence correlation spectroscopy (FCS). In the present study we show that FCS allowed us in a versatile and fast way to investigate the binding of Dervan polyamides to DNA. In particular it also shows its power in the quantitative detection of the formation of multimeric complexes and the in investigation of binding under nonphysiological conditions.