Nonfluorescent Molecules Research Articles

Heat action spectrum of Rhodamine B through two-photon absorption in near-infrared region studied by optical-probing photoacoustic spectroscopy

The heat action spectrum of Rhodamine B by two-photon absorption has been measured by a new system of optical-probing photoacoustic spectroscopy (OPPAS) coupled with a nanosecond near-infrared OPO laser. The highly resolved spectrum was obtained in the wide spectral range from 680 nm to 900 nm. The two-photon absorption cross-section of Rhodamine B was successfully determined by comparing the signal intensity of Rhodamine B with that of the reference. The OPPAS system was able to measure a well-resolved two-photon absorption spectrum of both fluorescent and non-fluorescent molecules in the wider spectral range from visible to near-infrared region.

Exploring Rotational Diffusion with Plasmonic Coupling.

Measuring the orientation dynamics of nanoparticles and nonfluorescent molecules in real time with optical methods is still a challenge in nanoscience and biochemistry. Here, we examine optoplasmonic sensing taking the rotational diffusion of plasmonic nanorods as an experimental model. Our detection method is based on monitoring the dark-field scattering of a relatively large sensor gold nanorod (GNR) (40 nm in diameter and 112 nm in length) as smaller plasmonic nanorods cross its near field. We observe the rotational motion of single small gold nanorods (three samples with about 5 nm in diameter and 15.5, 19.1, and 24.6 nm in length) in real time with a time resolution around 50 ns. Plasmonic coupling enhances the signal of the diffusing gold nanorods, which are 1 order of magnitude smaller in volume (about 300 nm3) than those used in our previous rotational diffusion experiments. We find a better angular sensitivity with plasmonic coupling in comparison to the free diffusion in the confocal volume. Yet, the angle sensitivity we find with plasmonic coupling is reduced compared to the sensitivity expected from simulations at fixed positions due to the simultaneous translational and rotational diffusion of the small nanorods. To get a reliable plasmonic sensor with the full angular sensitivity, it will be necessary to construct a plasmonic assembly with positions and orientations nearly fixed around the optimum geometry.

Facilitated Transport of EGFR Inhibitors Plays an Important Role in Their Cellular Uptake.

Epidermal growth factor receptor (EGFR) is a transmembrane protein commonly targeted by tyrosine kinase inhibitors (TKIs) as a front-line therapy for patients with many cancers including nonsmall cell lung cancer (NSCLC). Effective treatment requires efficient intracellular drug uptake and target binding. However, despite the recent success in the development of new TKI drugs, the mechanisms of uptake for many TKIs are still poorly understood due to the difficulty in imaging and measuring nonfluorescent drug molecules at a subcellular resolution. It has previously been shown that weakly basic TKI drugs are sequestered in lysosomes. Leveraging this property, we apply hyperspectral stimulated Raman scattering imaging to directly visualize and quantify two Food and Drug Administration-approved EGFR inhibitor drugs (lapatinib and afatinib) inside living cells and the changes in their cellular uptake upon the addition of organic cation transporter inhibitors. These single-cell quantitative measurements provide new insight into the role of membrane transporters in the uptake of TKI drugs in living cells.

Mapping electronic decoherence pathways in molecules.

Establishing the fundamental chemical principles that govern molecular electronic quantum decoherence has remained an outstanding challenge. Fundamental questions such as how solvent and intramolecular vibrations or chemical functionalization contribute to the decoherence remain unanswered and are beyond the reach of state-of-the-art theoretical and experimental approaches. Here we address this challenge by developing a strategy to isolate electronic decoherence pathways for molecular chromophores immersed in condensed phase environments that enables elucidating how electronic quantum coherence is lost. For this, we first identify resonance Raman spectroscopy as a general experimental method to reconstruct molecular spectral densities with full chemical complexity at room temperature, in solvent, and for fluorescent and non-fluorescent molecules. We then show how to quantitatively capture the decoherence dynamics from the spectral density and identify decoherence pathways by decomposing the overall coherence loss into contributions due to individual molecular vibrations and solvent modes. We illustrate the utility of the strategy by analyzing the electronic decoherence pathways of the DNA base thymine in water. Its electronic coherences decay in [Formula: see text]30 fs. The early-time decoherence is determined by intramolecular vibrations while the overall decay by solvent. Chemical substitution of thymine modulates the decoherence with hydrogen-bond interactions of the thymine ring with water leading to the fastest decoherence. Increasing temperature leads to faster decoherence as it enhances the importance of solvent contributions but leaves the early-time decoherence dynamics intact. The developed strategy opens key opportunities to establish the connection between molecular structure and quantum decoherence as needed to develop chemical strategies to rationally modulate it.

Development of fluorescence chemo sensor for selective histamine determination in spiked human plasma samples

Histamine is crucial for controlling a variety of physiological processes and its dysregulation is linked to various pathological conditions, including allergic disorders, autoimmune diseases and inflammatory conditions. Herein, a novel fluorescence chemo sensor was produced to measure histamine in the pure form and spiked human plasma matrix. The proposed method is based on chemical transformation of histamine into a fluorescent product, N-(2-(1H-imidazol-4-yl) ethyl)-2-bromoacetamide, exhibiting unique fluorescence properties compared to non-fluorescent histamine molecule. This transformation occurs through a sequence of chemical reactions involving the interaction of histamine with trimethylamine, resulting in the formation of a nucleophilic intermediate that subsequently reacts with electrophilic bromoacetyl bromide. The transformed fluorescent product demonstrates an emission at 340 nm after being excited at 250 nm. Significant concentration-dependent fluorescence enhancement was obtained enabling histamine determination. The procedures were examined for accuracy, precision, selectivity, and robustness in line with the ICH M10 recommendations. The method exhibits a lower limit of quantification at 0.25 ng/mL and dynamic detection throughout a linearity range of 1–200 ng/mL, providing accurate assessment of histamine in the plasma matrix.

Measuring Solvent Exchange in Silica Nanoparticles with Rotor-Based Fluorophore.

Measuring the diffusivity of molecules is the first step toward understanding their dependence and controlling diffusion, but the challenge increases with the decrease of molecular size, particularly for non-fluorescent and non-reactive molecules such as solvents. Here, the capability to monitor the solvent exchange process within the micropores of silica with millisecond time resolution is demonstrated, by simply embedding a rotor-based fluorophore (thioflavin T) in colloidal silica nanoparticles. Basically, the silica provides an extreme case of viscous microenvironment, which is affected by the polarity of the solvents. The fluorescence intensity traces can be well fitted to the Fickian diffusion model, allowing analytical solution of the diffusion process, and revealing the diffusion coefficients. The validation experiments, involving the water-to-ethanol and ethanol-to-water solvent exchange, the comparison of different drying conditions, and the variation in the degree of cross-linking in silica, confirmed the effectiveness and sensitivity of this method for characterizing diffusion in silica micropores. This work focuses on the method development of measuring diffusivity and the high temporal resolution in tracking solvent exchange dynamics over a short distance (within 165nm) opens enormous possibilities for further studies.

Molecular Accessibility and Diffusion of Resorufin in Zeolite Crystals.

We have used confocal laser scanning microscopy on the small, fluorescent resorufin dye molecule to visualize molecular accessibility and diffusion in the hierarchical, anisotropic pore structure of large (~10 μm-sized) zeolite-β crystals. The resorufin dye is widely used in life and materials science, but only in its deprotonated form because the protonated molecule is barely fluorescent in aqueous solution. In this work, we show that protonated resorufin is in fact strongly fluorescent when confined within zeolite micropores, thus enabling fluorescence microimaging experiments. We find that J-aggregation guest-guest interactions lead to a decrease in the measured fluorescence intensity that can be prevented by using non-fluorescent spacer molecules. We characterized the pore space by introducing resorufin from the outside solution and following its diffusion into zeolite-β crystals. The eventual homogeneous distribution of resorufin molecules throughout the zeolite indicates a fully accessible pore network. This enables the quantification of the diffusion coefficient in the straight pores of zeolite-β without the need for complex analysis, and we found a value of 3×10-15 m2 s-1 . Furthermore, we saw that diffusion through the straight pores of zeolite-β is impeded when crossing the boundaries between zeolite subunits.

Kinetic study of natural deep eutectic solvents by digital images: The importance of preparation time in sensing applications

The development of green solvents is a trending topic in both the industrial and the scientific community. In recent years, there has been a considerable interest in Deep Eutectic Solvents (DES) and Natural Deep Eutectic Solvents (NADES) since they are in agreement with Green Chemistry principles, their low cost, and also because of they are easy to prepare. However, no studies have been reported so far on the importance of preparation temperature and storage time of this kind of solvents.In this work, the preparation temperature, storage time and optical properties of a natural deep eutectic solvent composed of fructose, citric acid and water (1:1:5) were assayed by means of a kinetic study through digital images. The fluorescence response was monitored for at least 20 days, at preparation temperatures from 61 to 100 °C. Results showed, in most cases, an abrupt ascent and a linear decay of the signal whose slope depended on preparation temperature. 1H NMR techniques showed that no new C–C bonds were formed in NADES preparation and thus no chemical reaction was involved. Besides, by using 2D-NOESY, it was confirmed that the variation in NADES fluorescence cannot be attributed to changes in the spatial interaction modes of the eutectic system molecules.In order to demonstrate the scope of this study in analytical applications, the behavior of the eutectic system was tested with curcumin and neomycin as fluorescent and non-fluorescent molecules, respectively. The former showed a linear trend regardless of the time since preparation, while the latter only showed a linear response at short storage times.

Rapid live-cell sequential multiplexed imaging with fluorogenic RNAs.

Multiplexed detection of different cellular targets is an important goal in cell biology. However, multiplexed imaging in living cells is still extremely challenging. In terms of fluorescence imaging, significant spectral overlaps limit the number of distinguishable fluorophores that can be detected simultaneously. To solve this problem, in this project, we introduce a sequential imaging strategy termed “sequential Fluorogenic RNA Imaging-Enabled Sensor” (seqFRIES), to separately detect these spectrally overlapped fluorophores over time. Fluorogenic RNA aptamers are RNA strands that recognize and activate the fluorescence of otherwise non-fluorescent dye molecules. Here, we explored a list of reported fluorogenic RNA/dye pairs and identified five orthogonal ones. After genetically encoding all these fluorogenic RNAs together in cells, the corresponding dye molecules are added, imaged, and removed in a sequential manner. A very fast fluorescence activation and deactivation (in several minutes) was observed with these RNA/dye pairs. Taking advantages of such “image-and-wash” cycles, the multiplexing capacity of this imaging system is no longer limited by the excitation/emission spectral overlap of the fluorescent molecules. As a proof of concept, we show that the seqFRIES can perform robustly in both bacterial and mammalian cells, enabling multiplexed imaging of different RNAs, metabolites, and signaling molecules. We envision that this versatile multiplexed reporter system can be potentially adapted for imaging the cellular colocalization and dynamics of various target analytes, again inside living cells.

Super-resolution imaging of non-fluorescent molecules by photothermal relaxation localization microscopy

Super-resolution imaging of non-fluorescent molecules by photothermal relaxation localization microscopy

Palladium encapsulated mesoporous silica nanoparticles for the rapid detection of analytes.

We designed a simple, inexpensive, and user-friendly assay using mesoporous silica nanoparticles to detect analytes. Highly stable and uniform palladium nanoparticles covered with mesoporous silica (Pd@mSiO2) were generated and characterized extensively using physical methods. Human Serum Albumin (HSA) protein or ssDNA specific to the HIV gag region was capped onto the Pd@mSiO2 electrostatically. This "cap" prevented the Pd(0) inside the mesoporous silica nanoparticles from catalyzing the conversion of non-fluorescent molecules to fluorescent molecules. In the presence of target anti-HSA antibodies or complementary sequence (HIV gag), HSA protein or DNA cap dissociated from the surface of Pd@mSiO2-NH2 through the specific antigen-antibody reaction or DNA hybridization, allowing Pd(0) to convert the non-fluorescent molecules to fluorescent molecules. The limit and range of detection of anti-HSA antibodies were 3.8 nM and 3.8 nM to 133.3 nM, respectively. The limit and range of detection of HIV gag were 1.6 nM and 1.6 nM to 15 nM, respectively. This simple, inexpensive, "add sample and measure" diagnostic assay could potentially be incorporated into point of care diagnostics for low-resource settings.

Scytonemin redox status in a filamentous cyanobacterium visualized by an excitation-laser-line-scanning spontaneous Raman scattering spectral microscope

Some cyanobacteria produce a UVA-absorbing pigment, scytonemin, at extracellular sheaths. Although scytonemin-containing dark sheaths are recognizable through optical microscopes and its redox changes have been known for decades, there has been no report to obtain images of both oxidized and reduced scytonemins at subcellular resolution. Here, we show that a spontaneous Raman scattering spectral microscopy based on an excitation-laser-line-scanning method unveil 3D subcellular distributions of both the oxidized and reduced scytonemins in a filamentous cyanobacterium. The redox changes of scytonemin were supported by comparison in the Raman spectra between the cyanobacterial cells, solid-state scytonemin and quantum chemical normal mode analysis. Distributions of carotenoids, phycobilins, and the two redox forms of scytonemin were simultaneously visualized with an excitation wavelength at 1064 nm that is virtually free from the optical screening by the dark sheaths. The redox differentiation of scytonemin will advance our understanding of the redox homeostasis and secretion mechanisms of individual cyanobacteria as well as microscopic chemical environments in various microbial communities. The line-scanning Raman microscopy based on the 1064 nm excitation is thus a promising tool for exploring hitherto unreported Raman spectral features and distribution of nonfluorescent molecules embedded below nontransparent layers for visible light, while avoiding interference by autofluorescence.

344.3: Extracellular Vesicles Released During Normothermic Machine Perfusion Are Associated With Human Donor Kidney Characteristics

Background: Extracellular Vesicles (EVs) represent stable, tissue specific nano-sized particles that reflect the conditional state of their tissue of origin. Here, the dynamic release and phenotype of kidney EVs was characterized and quantified during Normothermic Machine Perfusion (NMP) of Expanded-Criteria Donor (ECD) kidneys to examine whether EVs could function as a potential biomarker for assessing kidney quality before transplantation. Materials & Methods: Eight discarded ECD kidneys (~13 ± 5 hours of cold ischemia, age 68 ± 7 (mean ± standard deviation), all male) were perfused in a closed system at 37 0C for 6 hours. Perfusates were taken before and at 1, 3 and 6 hours of NMP and examined with Nanoparticle Tracking Analysis (NTA) and Imaging Flow Cytometry (IFCM). For IFCM, perfusates were stained with the tetraspanins CD9, CD63 or CD81 (general EV markers), or a mix of these three markers in combination with CFDA-SE (a non-fluorescent molecule that acquires fluorescent properties after cleavage by intravesicular esterases) to identify, quantify and characterize EVs. Results: Analysis of perfusates with NTA revealed that the majority of nanoparticles present in the perfusates are <300 nm. For CFSE and the mix of tetraspanin double-positive EVs, we observed a ~700/ 740/ 560 fold increase compared to EV levels before perfusion at 1, 3 and 6 hours of NMP, respectively. Analysis of EV concentrations with crude donor characteristics (e.g. age, cold ischemia time (CIT), kidney weight) and NMP viability characteristics (renal flow, renal flow resistance, urine production) revealed that double-positive EV are negatively correlated with CIT whilst positive correlations were found with donor age after the first hour of NMP. Furthermore, tetraspanin CD81 was found to represent the majority (~75%) of the excreted double-positive EV (CD9: ~16%/ CD63 ~8%) (Figure). Conclusion: EVs <300 nm are released by ECD kidneys during NMP with highest excretion levels during the first hour of perfusion. Tetraspanin CD81 is predominantly present on these EVs, and EV concentrations were shown to be correlated with well-established indicators of kidney quality such as donor age and CIT. The characterization of the excreted EVs as well as their correlation with clinical parameters provide a starting point to study their role as potential biomarkers of kidney quality.

Photothermal spectroscopy and micro/nanofluidics

Photothermal spectroscopy, a remarkable detection method that can analyze microscale objects in a noninvasive and nondestructive manner, has been successfully coupled with micro/nanofluidic devices. Specifically, methods that employ a thermal lens microscope (TLM), including a photothermal optical phase shift and photothermal optical diffraction, are a powerful tool for the sensitive detection of nonfluorescent or nonlabeled molecules in micro/nanofluidic channels. This review focuses on the family of TLMs in terms of their historical development. Their recent applications, ranging from separation, particle, biomedical, energy, and environmental analyses, are summarized, and future perspectives in nanoscale liquid science, system integration, and biological studies, such as single-cell analyses, are also discussed.

Detection of SARS-CoV-2 RNA Using a DNA Aptamer Mimic of Green Fluorescent Protein.

RNA detection is important in diverse diagnostic and analytical applications. RNAs can be rapidly detected using molecular beacons, which fluoresce upon hybridizing to a target RNA but require oligonucleotides with complex fluorescent dye and quencher conjugations. Here, we describe a simplified method for rapid fluorescence detection of a target RNA using simple unmodified DNA oligonucleotides. To detect RNA, we developed Lettuce, a fluorogenic DNA aptamer that binds and activates the fluorescence of DFHBI-1T, an otherwise nonfluorescent molecule that resembles the chromophore found in green fluorescent protein. Lettuce was selected from a randomized DNA library based on binding to DFHBI-agarose. We further show that Lettuce can be split into two separate oligonucleotide components, which are nonfluorescent on their own but become fluorescent when their proximity is induced by a target RNA. We designed several pairs of split Lettuce fragments that contain an additional 15-20 nucleotides that are complementary to adjacent regions of the SARS-CoV-2 RNA, resulting in Lettuce fluorescence only in the presence of the viral RNA. Overall, these studies describe a simplified RNA detection approach using fully unmodified DNA oligonucleotides that reconstitute the Lettuce aptamer templated by RNA.

Transient absorption spectroscopy and imaging of redox in muscle mitochondria.

Mitochondrial redox is an important indicator of cell metabolism and health, with implications in cancer, diabetes, aging, neurodegenerative diseases, and mitochondrial disease. The most common method to observe redox of individual cells and mitochondria is through fluorescence of NADH and FAD+, endogenous cofactors serve as electron transport inputs to the mitochondrial respiratory chain. Yet this leaves out redox within the respiratory chain itself. To a degree, the missing information can be filled in by exogenous fluorophores, but at the risk of disturbed mitochondrial permeability and respiration. Here we show that variations in respiratory chain redox can be detected up by visible-wavelength transient absorption microscopy (TAM). In TAM, the selection of pump and probe wavelengths can provide multiphoton imaging contrast between non-fluorescent molecules. Here, we applied TAM with a pump at 520nm and probe at 450nm, 490nm, and 620nm to elicit redox contrast from mitochondrial respiratory chain hemeproteins. Experiments were performed with reduced and oxidized preparations of isolated mitochondria and whole muscle fibers, using mitochondrial fuels (malate, pyruvate, and succinate) to set up physiologically relevant oxidation levels. TAM images of muscle fibers were analyzed with multivariate curve resolution (MCR), revealing that the response at 620nm probe provides the best redox contrast and the most consistent response between whole cells and isolated mitochondria.

Rapid and sensitive detection of H2S by a 4-phenylselenium coumarin as a dual-active-site fluorescent probe

Hydrogen sulfide (H2S) is associated with numerous psychological and pathological processes, and related closely with human health and diseases. The detection of H2S is a key to better understand its roles. In this paper, we developed a 4-phenylselenium coumarin (PhSeCou) as a rapid-response fluorescent probe for the selective and sensitive detection of H2S. The probe PhSeCou is a stable and almost non-fluorescent molecule in a PBS buffer, and it can rapidly (< 60 s) sense H2S to give a strongly fluorescent product, Cou-H2S (Φf = 0.11, centered at 563 nm), thereby achieving to a sensitive detection at an ultra-low limit of detection (8.8 nM). The fluorescent probe displays a high selectivity for H2S over biothiols and other relevant analytes. What is more, through fluorescence cell-imaging, PhSeCou was successfully applied to detect exo- and endogenous H2S in living cells.

Single-Particle Catalytic Analysis by a Photon Burst Counting Technique Combined with a Microfluidic Chip.

Single-particle catalytic analysis plays an important role to understand the catalytic mechanism of nanocatalysts. Currently, some methods are used to study the relationship between single-particle catalytic activity and morphology. However, there is still lack of a simple and rapid analysis method for evaluating the catalytic activity of an individual nanocatalyst that freely moves in solution. Here, we proposed a novel single-particle catalytic analysis method for investigating the catalytic activity of a free nanocatalyst. Its working principle is based on the photon burst counting analysis on fluorescent catalytic products of an individual nanocatalyst combined with a microfluidic chip. In this study, we used the reduction reaction of resazurin (RZ) to resorufin (RF) catalyzed by gold nanoparticles (GNPs) as a model. When nonfluorescent RZ molecules in one microchannel of the microfluidic chip mixed with the GNPs flowing in another channel under the control of flow rates, each individual photon burst from the catalytic product RF by GNPs was measured in real time with a constructed flow single-particle catalytic analysis (SPCA) system. With the method, the obtained intensity of each photon burst reflects the capacity of a particle to catalyze RZ molecules into RF(s). The number of photon burst within sampling time reflects the particle number of GNPs with catalytic activity. The experimental conditions including the mixing mode of the nanocatalyst and the substrate, the flow rate, RZ concentration, and detection time were optimized. Finally, the method was successfully used to study the catalytic activity of GNPs with different sizes and morphologies.

Visualising G-quadruplex DNA dynamics in live cells by fluorescence lifetime imaging microscopy

Guanine rich regions of oligonucleotides fold into quadruple-stranded structures called G-quadruplexes (G4s). Increasing evidence suggests that these G4 structures form in vivo and play a crucial role in cellular processes. However, their direct observation in live cells remains a challenge. Here we demonstrate that a fluorescent probe (DAOTA-M2) in conjunction with fluorescence lifetime imaging microscopy (FLIM) can identify G4s within nuclei of live and fixed cells. We present a FLIM-based cellular assay to study the interaction of non-fluorescent small molecules with G4s and apply it to a wide range of drug candidates. We also demonstrate that DAOTA-M2 can be used to study G4 stability in live cells. Reduction of FancJ and RTEL1 expression in mammalian cells increases the DAOTA-M2 lifetime and therefore suggests an increased number of G4s in these cells, implying that FancJ and RTEL1 play a role in resolving G4 structures in cellulo.

Microfluidic platform enables tailored translocation and reaction cascades in nanoliter droplet networks

In the field of bottom-up synthetic biology, lipid membranes are the scaffold to create minimal cells and mimic reactions and processes at or across the membrane. In this context, we employ here a versatile microfluidic platform that enables precise positioning of nanoliter droplets with user-specified lipid compositions and in a defined pattern. Adjacent droplets make contact and form a droplet interface bilayer to simulate cellular membranes. Translocation of molecules across membranes are tailored by the addition of alpha-hemolysin to selected droplets. Moreover, we developed a protocol to analyze the translocation of non-fluorescent molecules between droplets with mass spectrometry. Our method is capable of automated formation of one- and two-dimensional droplet networks, which we demonstrated by connecting droplets containing different compound and enzyme solutions to perform translocation experiments and a multistep enzymatic cascade reaction across the droplet network. Our platform opens doors for creating complex artificial systems for bottom-up synthetic biology.