The pros and cons of nucleic acid-amplified immunoassays-a comparative study on the quantitation of prostate-specific antigen with and without rolling circle amplification.

Integrating isothermal nucleic acid amplification strategies into immunoassays can significantly decrease analytical limits of detection (LODs). On the other hand, an amplification step adds time, complication, reagents, and costs to the assay format. To evaluate the pros and cons in the context of heterogeneous multistep immunoassays, we quantified prostate-specific antigen (PSA) with and without rolling circle amplification (RCA). In addition, we compared time-gated (TG) with continuous-wave (CW) photoluminescence (PL) detection using a terbium complex and a fluorescein dye, respectively. For both direct (non-amplified) and amplified assays, TG PL detection provided circa four- to eightfold lower LODs, illustrating the importance of autofluorescence background suppression even for multi-wash assay formats. Amplified assays required an approximately 2.4 h longer assay time but led to almost 100-fold lower LODs down to 1.3 pg/mL of PSA. Implementation of TG-FRET (using a Tb-Cy5.5 donor-acceptor pair) into the RCA immunoassay resulted in a slightly higher LOD (3.0 pg/mL), but the ratiometric detection format provided important benefits, such as higher reproducibility, lower standard deviations, and multiplexing capability. Overall, our direct comparison demonstrated the importance of biological background suppression even in heterogeneous assays and the potential of using isothermal RCA for strongly decreasing analytical LODs, making such assays viable alternatives to conventional enzyme-linked immunosorbent assays (ELISAs).

Mercury-induced excitotoxicity in presynaptic brain nerve terminals: modulatory effects of carbonaceous airborne particulate simulants.

Multipollutant approach is a breakthrough in up-to-date environmental quality and health risk estimation. Both mercury and carbonaceous air particulate are hazardous neurotoxicants. Here, the ability of carbonaceous air particulate simulants, i.e. carbon dots obtained by heating of organics, and nanodiamonds, to influence Hg2+-induced neurotoxicity was monitored using biological system, i.e. presynaptic rat cortex nerve terminals. Using HgCl2 and classical reducing/chelating agents, an adequate synaptic parameter, i.e. the extracellular level of key excitatory neurotransmitter L-[14C]glutamate, was selected for further analysis. HgCl2 starting from 5 µM caused an acute and concentration-dependent increase in the extracellular L-[14C]glutamate level in nerve terminals. Combined application of Hg2+ and carbon dots from heating of citric acid/urea showed that this simulant was able to mitigate in an acute manner excitotoxic Hg2+-induced increase in the extracellular L-[14C]glutamate level in nerve terminals by 37%. These carbon dots and Hg2+ acted as a complex in nerve terminals that was confirmed with fluorimetric data on Hg2+-induced changes in their spectroscopic features. Nanodiamonds and carbon dots from ß-alanine were not able to mitigate a Hg2+-induced increase in the extracellular L-[14C]glutamate level in nerve terminals. Developed approach can be applicable for monitoring capability of different particles/compounds to have Hg2+-chelating signs in the biological systems. Therefore, among testing simulants, the only carbon dots from citric acid/urea were able to mitigate acute Hg2+-induced neurotoxicity in nerve terminals, thereby showing a variety of effects of carbonaceous airborne particulate in situ and its potential to interfere and modulate Hg2+-associated health hazard.

Discrimination of the V600E Mutation in BRAF by Rolling Circle Amplification and Förster Resonance Energy Transfer.

The quantification of very low concentrations of circulating tumor DNA (ctDNA) biomarkers from liquid biopsies has become an important requirement for clinical diagnostics and personalized medicine. In particular, the simultaneous detection of wild-type (WT) dsDNA and their cancer-related counterparts presenting single-point mutations with simple, sensitive, specific, and reproducible technologies is paramount for ctDNA assays in clinical practice. Here, we present the development and evaluation of an amplified dsDNA assay based on a combination of isothermal rolling circle amplification (RCA) and time-gated Förster resonance energy transfer (TG-FRET) between a Tb donor and two dye (Cy3.5 and Cy5.5) acceptors. The RCA-FRET assay is free of washing and separation steps and can quantify both WT and mutated (MT) (V600E) dsDNA in the BRAF gene from a single sample in the 75 fM to 4.5 pM (4.5 × 105 to 2.7 × 107 copies) concentration range. This assay includes all steps from denaturation of the dsDNA targets to the final duplexed quantification of WT and MT targets. High assay performance at different dsDNA sequence lengths and high target specificity even in the presence of a large excess of nonspecific cell-free DNA from human plasma samples demonstrated the applicability to clinical samples. The RCA-FRET single-point mutation sensor has the potential to become an important complementary technique for analyzing liquid biopsies in advanced cancer diagnostics.

Insight into the mechanism of the photoluminescence of carbon nanoparticles derived from cryogenic studies.

The unexpected discovery of the photoluminescence of carbon nanoparticles attracted the attention of many researchers and resulted in their use in a variety of applications. However, the origin of their emission is still obscure, and the majority of the discussions on the subject focus on their molecular and/or excitonic emissive states. We performed cryogenic studies down to 10 K and did not observe any signatures of suppressed molecular relaxation - the spectra remained broad, showing large unaltered Stokes shifts and temperature-independent emission intensities and lifetimes below 80 K with a weak dependence above this value. We demonstrated that the most general features of carbon nanoparticles, the very large Stokes shifts and considerable differences between the absorption and excitation spectra, are the result of the formation of a dynamic defect, the self-trapped Frenkel exciton. It looks like the distorted domain of the H-aggregate due to the exciton-lattice interaction and the local overheating caused by the exciton relaxation. In addition, at low temperatures the long-lifetime spectral component was found and was attributed to phosphorescence. The obtained results strongly support the excitonic nature of the fluorescence of nanocarbon materials.

Harmful impact on presynaptic glutamate and GABA transport by carbon dots synthesized from sulfur-containing carbohydrate precursor.

Carbon nanoparticles that may be potent air pollutants with adverse effects on human health often contain heteroatoms including sulfur. In order to study in detail their effects on different physiological and biochemical processes, artificially produced carbon dots (CDs) with well-controlled composition that allows fluorescence detection may be of great use. Having been prepared from different types of organic precursors, CDs expose different atoms at their surface suggesting a broad variation of functional groups. Recently, we demonstrated neurotoxic properties of CDs synthesized from the amino acid ß-alanine, and it is of importance to analyze whether CDs obtained from different precursors and particularly those exposing sulfur atoms induce similar neurotoxic effects. This study focused on synthesis of CDs from the sulfur-containing precursor thiourea-CDs (TU-CDs) with a size less than 10 nm, their characterization, and neuroactivity assessment. Neuroactive properties of TU-CDs were analyzed based on their effects on the key characteristics of glutamatergic and γ-aminobutyric acid (GABA) neurotransmission in isolated rat brain nerve terminals. It was observed that TU-CDs (0.5-1.0 mg/ml) attenuated the initial velocity of Na+-dependent transporter-mediated uptake and accumulation of L-[14C]glutamate and [3H]GABA by nerve terminals in a dose-dependent manner and increased the ambient level of the neurotransmitters. Starting from the concentration of 0.2 mg/ml, TU-CDs evoked a gradual dose-dependent depolarization of the plasma membrane of nerve terminals measured with the cationic potentiometric dye rhodamine 6G. Within the concentration range of 0.1-0.5 mg/ml, TU-CDs caused an "unphysiological" step-like increase in fluorescence intensity of the рН-sensitive fluorescent dye acridine orange accumulated by synaptic vesicles. Therefore, despite different surface properties and fluorescent features of CDs prepared from different starting materials (thiourea and ß-alanine), their principal neurotoxic effects are analogous but displayed at a different level of efficiency. Sulfur-containing TU-CDs exhibit lower effects (by ~30%) on glutamate and GABA transport in the nerve terminals in comparison with sulfur-free ß-alanine CDs. Our results suggest considering that an uncontrolled presence of carbon-containing particulate matter in the human environment may pose a toxicity risk for the central nervous system.

The origin of emissive states of carbon nanoparticles derived from ensemble-averaged and single-molecular studies.

At present, there is no consensus understanding on the origin of photoluminescence of carbon nanoparticles, particularly the so-called carbon dots. Providing comparative analysis of spectroscopic studies in solution and on a single-molecular level, we demonstrate that these particles behave collectively as fixed single dipoles and probably are the quantum emitter entities. Their spectral and lifetime heterogeneity in solutions is explained by variation of the local chemical environment within and around luminescence centers. Hence, the carbon dots possess a unique hybrid combination of fluorescence properties peculiar to dye molecules, their conjugates and semiconductor nanocrystals. It is proposed that their optical properties are due to generation of H-aggregate-type excitonic states with their coherence spreading over the whole nanoparticles.

Super-Resolution Optical Fluctuation Bio-Imaging with Dual-Color Carbon Nanodots.

Success in super-resolution imaging relies on a proper choice of fluorescent probes. Here, we suggest novel easily produced and biocompatible nanoparticles-carbon nanodots-for super-resolution optical fluctuation bioimaging (SOFI). The particles revealed an intrinsic dual-color fluorescence, which corresponds to two subpopulations of particles of different electric charges. The neutral nanoparticles localize to cellular nuclei suggesting their potential use as an inexpensive, easily produced nucleus-specific label. The single particle study revealed that the carbon nanodots possess a unique hybrid combination of fluorescence properties exhibiting characteristics of both dye molecules and semiconductor nanocrystals. The results suggest that charge trapping and redistribution on the surface of the particles triggers their transitions between emissive and dark states. These findings open up new possibilities for the utilization of carbon nanodots in the various super-resolution microscopy methods based on stochastic optical switching.

Visualization and detection of live and apoptotic cells with fluorescent carbon nanoparticles.

Apoptosis is a genetically encoded cell death program that involves different processes occurring on molecular and sub-cellular levels. Here we report on its new features--the increased accumulation of fluorescent carbon nanoparticles (CDots) in cells and their changed distribution within cell interior, which can witness on altered mechanisms of their translocation through the membrane. The comparative studies of living (intact) and apoptotic cells were provided with two cell lines (HeLa, Vero) using two types of fluorescent nanoparticles ("violet" and "blue" CDots). In all studied cases the images of living and apoptotic cells were different; the apoptotic cells incorporated larger number of CDots resulting in their much brighter images. These nanoparticles are distributed in cell cytoplasm, however, when the cells are fixed and treated with detergent, their nucleus is also labeled. Flow cytometry allows distinguishing the sub-populations of living and apoptotic cells in their cultures and suggests a very cheap and easy way to characterize them.

Neuromodulatory properties of fluorescent carbon dots: effect on exocytotic release, uptake and ambient level of glutamate and GABA in brain nerve terminals.

Carbon dots (C-dots), a recently discovered class of fluorescent nano-sized particles with pure carbon core, have great bioanalytical potential. Neuroactive properties of fluorescent C-dots obtained from ß-alanine by microwave heating were assessed based on the analysis of their effects on the key characteristics of GABA- and glutamatergic neurotransmission in isolated rat brain nerve terminals. It was found that C-dots (40-800 µg/ml) in dose-dependent manner: (1) decreased exocytotic release of [(3)H]GABA and L-[(14)C]glutamate; (2) reduced acidification of synaptic vesicles; (3) attenuated the initial velocity of Na(+)-dependent transporter-mediated uptake of [(3)H]GABA and L-[(14)C]glutamate; (4) increased the ambient level of the neurotransmitters, nevertheless (5) did not change significantly the potential of the plasma membrane of nerve terminals. Almost complete suppression of exocytotic release of the neurotransmitters was caused by C-dots at a concentration of 800 µg/ml. Fluorescent and neuromodulatory features combined in C-dots create base for their potential usage for labeling and visualization of key processes in nerve terminals, and also in theranostics. In addition, natural presence of carbon-containing nanoparticles in the human food chain and in the air may provoke the development of neurologic consequences.

Photoluminescence of carbon nanodots: dipole emission centers and electron-phonon coupling.

Inorganic carbon nanomaterials, also called carbon nanodots, exhibit a strong photoluminescence with unusual properties and, thus, have been the focus of intense research. Nonetheless, the origin of their photoluminescence is still unclear and the subject of scientific debates. Here, we present a single particle comprehensive study of carbon nanodot photoluminescence, which combines emission and lifetime spectroscopy, defocused emission dipole imaging, azimuthally polarized excitation dipole scanning, nanocavity-based quantum yield measurements, high resolution transmission electron microscopy, and atomic force microscopy. We find that photoluminescent carbon nanodots behave as electric dipoles, both in absorption and emission, and that their emission originates from the recombination of photogenerated charges on defect centers involving a strong coupling between the electronic transition and collective vibrations of the lattice structure.

Fluorescent carbon nanomaterials: "quantum dots" or nanoclusters?

Despite many efforts, the mechanisms of light absorption and emission of small fluorescent carbon nanoparticles (C-dots) are still unresolved and are a subject of active discussion. In this work we address the question as to whether the fluorescence is a collective property of these nanoparticles or they are composed of assembled individual emitters. Selecting three types of C-dots with "violet", "blue" and "green" emissions and performing a detailed study of fluorescence intensity, lifetime and time-resolved anisotropy as a function of excitation and emission wavelengths together with the effect of viscogen and dynamic fluorescence quencher, we demonstrate that the C-dots represent assemblies of surface-exposed fluorophores. They behave as individual emitters, display electronic anisotropy, do not exchange their excited-state energies via homo-FRET and possibly display sub-nanosecond intra-particle mobility.

Novel fluorescent carbonic nanomaterials for sensing and imaging.

Small brightly fluorescent carbon nanoparticles have emerged as a new class of materials important for sensing and imaging applications. We analyze comparatively the properties of nanodiamonds, graphene and graphene oxide 'dots', of modified carbon nanotubes and of diverse carbon nanoparticles known as 'C-dots' obtained by different methods. The mechanisms of their light absorption and luminescence emission are still unresolved and the arguments are presented for their common origin. Regarding present and potential applications, we provide critical comparison with the other types of fluorescence reporters, such as organic dyes and semiconductor quantum dots. Their most prospective applications in sensing (based on the changes of intensity, FRET and lifetime) and in imaging technologies on the level of living cells and whole bodies are overviewed. The possibilities for design on their basis of multifunctional nanocomposites on a broader scale of theranostics are outlined.