Dye Pair Research Articles

Nanofiltration Membranes Containing a Metal-Polyphenol Network Layer: Using Casting Solution pH as a Tool to Tailor the Separation Performance.

Thin-film composite (TFC) membranes containing metal-polyphenol network (MPN) selective layers were fabricated using a supramolecular self-assembly between tannic acid (TA) and ferric ion (Fe3+). The TA-Fe3+ thin film was coated on a porous polyacrylonitrile support using aqueous solutions of TA and FeCl3 via a layer-by-layer deposition technique. The pH of the TA solution was used as a tool to alter the membrane characteristics. The surface porosity and water contact angle of the fabricated membranes gradually decreased as the pH of TA casting solutions was increased from 3 to 8.5 for both single-layered and double-layered TA-Fe3+ TFC membranes. This allowed us to tune the water permeance and the retentions of water-soluble neutral and anionic molecules by the MPN membranes by varying the pH of the casting solution. It has been shown that the water permeance decreased from 184 to 156 L·m-2·h-1·bar-1 for single TA-Fe3+ layer coated membranes when the pH was increased from 3 to 8.5, while it declined from 51 to 17 for the double TA-Fe3+ layer. Anionic solutes in aqueous solutions were highly retained compared to neutral components as the TFC membranes had a negative surface charge. Retentions of 95 and 90% were achieved for naphthol green B and orange II dyes by a double-layered M4 membrane fabricated at pH 8.5, while only 13% retention was found for the neutral riboflavin. The neutral dye riboflavin permeated 30.8 times higher than the anionic dye naphthol green B during a mixed dye filtration test through the TFC membrane prepared by using a TA solution of pH 8.5. To the best of our knowledge, this is the highest selectivity of a neutral/anionic dye pair so far reported for a TFC membrane having an MPN selective layer. Moreover, fouling tests have demonstrated that the MPN separation layers exhibit robust stability and adequate antifouling performance with a flux recovery ratio as high as 82%.

Long wavelength near-infrared and red light-driven consecutive photo-induced electron transfer for highly effective photoredox catalysis

Consecutive photoinduced electron transfer (conPET) processes accumulate the energies of two photons to overcome the thermodynamic limit of traditional photoredox catalysis. However, the excitation wavelength of conPET systems mainly focused on short wavelength visible light, leading to photodamage and incompatibility with large-scale reactions. Herein, we report on conPET systems triggered by near-infrared (NIR) and red light. Specifically, a blue-absorbing conPET photocatalyst, perylene diimide (PDI) is sensitized by a palladium-based photosensitizer to triplet excited state (3PDI*), which generates PDI radical anion (PDI•–) over 100-fold faster than that in the conventional conPET. Accordingly, photoreduction with superior reaction rate and penetration depth, as well as reduced photodamage is detected. More importantly, our work offers comprehensive design rules for the triplet-mediated conPET strategy, whose versatility is confirmed by metal-free dye pairs and NIR-active PtTNP/PDI. Notably, our work achieves NIR-driven atom transfer radical polymerization using an inert aromatic halide as the initiator.

An Aggregation-Induced Emission-Based Dual Emitting Nanoprobe for Detecting Intracellular pH and Unravelling Metabolic Variations in Differentiating Lymphocytes.

Monitoring T lymphocyte differentiation is essential for understanding T cell fate regulation and advancing adoptive T cell immunotherapy. However, current biomarker analysis methods necessitate cell lysis, leading to source depletion. Intracellular pH (pHi) can be affected by the presence of lactic acid (LA), a metabolic mediator of T cell activity such as glycolysis during T cell activation; therefore, it is a potentially a good biomarker of T cell state. In this work, a dual emitting enhancement-based nanoprobe, namely, AIEgen@F127-AptCD8, was developed to accurately detect the pHi of T cells to "read" the T cell differentiation process. The nanocore of this probe comprises a pair of AIE dyes, TPE-AMC (pH-sensitive moiety) and TPE-TCF, that form a donor-acceptor pair for sensitive detection of pHi by dual emitting enhancement analysis. The nanoprobe exhibits a distinctly sensitive narrow range of pHi values (from 6.0 to 7.4) that can precisely distinguish the differentiated lymphocytes from naïve ones based on their distinct pHi profiles. Activated CD8+ T cells demonstrate lower pHi (6.49 ± 0.09) than the naïve cells (7.26 ± 0.11); Jurkat cells exhibit lower pHi (6.43 ± 0.06) compared to that of nonactivated ones (7.29 ± 0.09) on 7 days post-activation. The glycolytic product profiles in T cells strongly correlate with their pHi profiles, ascertaining the reliability of probing pHi for predicting T cell states. The specificity and dynamic detection capabilities of this nanoprobe make it a promising tool for indirectly and noninvasively monitoring T cell activation and differentiation states.

Sensing of Gunshot Residue components from real sample using Fluorescence Resonance Energy Transfer

The present work represents a Fluorescence Resonance Energy Transfer (FRET) based sensing method for detecting Gunshot Residue (GSR) components. Two laser dyes Acf and RhB have been used as donor and acceptor respectively in the FRET pair. The real sample was collected after test firing in a forensic science laboratory. On the other hand, a standard GSR solution has been prepared in the laboratory. For the preparation of standard GSR solutions, we used the water solutions of the salts BaCl2, SbCl3, and Pb(NO3)2. The FRET efficiency was measured between Acf and RhB to sense the presence of GSR components (Pb+2, Ba+2, and Sb+3) in both real sample and standard solution by mixing the salts in aqueous solution. It has been observed that the FRET efficiency systematically decreases in the presence of GSR components. To amplify the FRET efficiency of the dye pair, inorganic clay dispersion (laponite) was used. The enhancement in FRET efficiency represents a better sensitivity of the proposed sensor. The current sensor is useful for the quantification of concentrations of the GSR components in a real sample.

Click Chemistry with Cell-Permeable Fluorophores Expands the Choice of Bioorthogonal Markers for Two-Color Live-Cell STED Nanoscopy.

STED nanoscopy allows for the direct observation of dynamic processes in living cells and tissues with diffraction-unlimited resolution. Although fluorescent proteins can be used for STED imaging, these labels are often outperformed in photostability by organic fluorescent dyes. This feature is especially crucial for time-lapse imaging. Unlike fluorescent proteins, organic fluorophores cannot be genetically fused to a target protein but require different labeling strategies. To achieve simultaneous imaging of more than one protein in the interior of the cell with organic fluorophores, bioorthogonal labeling techniques and cell-permeable dyes are needed. In addition, the fluorophores should preferentially emit in the red spectral range to reduce the potential phototoxic effects that can be induced by the STED light, which further restricts the choice of suitable markers. In this work, we selected five different cell-permeable organic dyes that fulfill all of the above requirements and applied them for SPIEDAC click labeling inside living cells. By combining click-chemistry-based protein labeling with other orthogonal and highly specific labeling methods, we demonstrate two-color STED imaging of different target structures in living specimens using different dye pairs. The excellent photostability of the dyes enables STED imaging for up to 60 frames, allowing the observation of dynamic processes in living cells over extended time periods at super-resolution.

Investigating the Effect of RNA Scaffolds on the Multicolor Fluorogenic Aptamer Pepper in Different Bacterial Species.

RNA synthetic biology tools have primarily been applied in E. coli; however, many other bacteria are of industrial and clinical significance. Thus, the multicolor fluorogenic aptamer Pepper was evaluated in both Gram-positive and Gram-negative bacteria. Suitable HBC-Pepper dye pairs were identified that give blue, green, or red fluorescence signals in the E. coli, Bacillus subtilis, and Salmonella enterica serovar Typhimurium (S. Typhimurium). Furthermore, we found that different RNA scaffolds have a drastic effect on in vivo fluorescence, which did not correlate with the in vitro folding efficiency. One such scaffold termed DF30-tRNA displays 199-fold greater fluorescence than the Pepper aptamer alone and permits simultaneous dual color imaging in live cells.

Strain-Promoted Cycloadditions in Lipid Bilayers Triggered by Liposome Fusion.

Due to the variety of roles served by the cell membrane, its composition and structure are complex, making it difficult to study. Bioorthogonal reactions, such as the strain promoted azide-alkyne cycloaddition (SPAAC), are powerful tools for exploring the function of biomolecules in their native environment but have been largely unexplored within the context of lipid bilayers. Here, we developed a new approach to study the SPAAC reaction in liposomal membranes using azide- and strained alkyne-functionalized Förster resonance energy transfer (FRET) dye pairs. This study represents the first characterization of the SPAAC reaction between diffusing molecules inside liposomal membranes. Potential applications of this work include in situ bioorthogonal labeling of membrane proteins, improved understanding of membrane dynamics and fluidity, and the generation of new probes for biosensing assays.

Strain‐Promoted Cycloadditions in Lipid Bilayers Triggered by Liposome Fusion

AbstractDue to the variety of roles served by the cell membrane, its composition and structure are complex, making it difficult to study. Bioorthogonal reactions, such as the strain promoted azide‐alkyne cycloaddition (SPAAC), are powerful tools for exploring the function of biomolecules in their native environment but have been largely unexplored within the context of lipid bilayers. Here, we developed a new approach to study the SPAAC reaction in liposomal membranes using azide‐ and strained alkyne‐functionalized Förster resonance energy transfer (FRET) dye pairs. This study represents the first characterization of the SPAAC reaction between diffusing molecules inside liposomal membranes. Potential applications of this work include in situ bioorthogonal labeling of membrane proteins, improved understanding of membrane dynamics and fluidity, and the generation of new probes for biosensing assays.

Control of the fluorescence lifetime in dye based nanoparticles.

Fluorescent dye based nanoparticles (NPs) have received increased interest due to their high brightness and stability. In fluorescence microscopy and assays, high signal to background ratios and multiple channels of detection are highly coveted. To this end, time-resolved imaging offers suppression of background and temporal separation of spectrally overlapping signals. Although dye based NPs and time-resolved imaging are widely used individually, the combination of the two is uncommon. This is likely due to that dye based NPs in general display shortened and non-mono-exponential lifetimes. The lower quality of the lifetime signal from dyes in NPs is caused by aggregation caused quenching (ACQ) and energy migration to dark states in NPs. Here, we report a solution to this problem by the use of the small-molecule ionic isolation lattices (SMILES) concept to prevent ACQ. Additionally, incorporation of FRET pairs of dyes locks the exciton on the FRET acceptor providing control of the fluorescence lifetime. We demonstrate how SMILES NPs with a few percent rhodamine and diazaoxatriangulenium FRET acceptors imbedded with a cyanine donor dye give identical emission spectra and high quantum yields but very different fluorescence lifetimes of 3 ns and 26 ns, respectively. The two spectrally identical NPs are easily distinguished at the single particle level in fluorescence lifetime imaging. The doping approach for dye based NPs provides predictable fluorescence lifetimes and allows for these bright imaging reagents to be used in time-resolved imaging detection modalities.

Linear spectral unmixing analysis in single-molecule FRET spectroscopy for fluorophores with large spectral overlap.

Fluorescence resonance energy transfer (FRET) is a highly useful tool to investigate biomolecular interactions and dynamics in single-molecule spectroscopy and nanoscopy. However, the use of spectrally overlapping dye pairs results in various artifact signals that prevent accurate determination of FRET values. In this paper, an algorithmic method of spectral unmixing was devised to extract FRET values of spectrally overlapping dye pairs at the single molecule level. Application of this method allows the determination of both the donor-acceptor composition and the FRET efficiency of the samples labelled with spectrally overlapping dye pairs.

Development of a peptide microarray-based metal-enhanced fluorescence assay for ultrasensitive detection of multiple matrix metalloproteinase activities by using a gold nanorod-polymer substrate

Matrix metalloproteinases (MMPs) are attractive biomarkers for cancer diagnosis and treatment, while it is still a challenge to precise analysis of MMP activities owing to their very low abundance in the biological samples, especially at the early stages of tumors. Herein, a peptide microarray-based metal-enhanced fluorescence assay (PMMEFA) is proposed to simultaneously detect MMP-1, -2, -3, -7, -9, and -13 activities. The assay involves immobilization of Förster resonance energy transfer dye pair decorated peptides (FRET-peptides) on a poly(glycidyl methacrylate-co-2-hydroxyethyl methacrylate) coated gold nanorod modified glass slide (GNR@P(GMA-HEMA)). To fabricate the GNR@P(GMA-HEMA) slide, GNRs are self-assembled onto an aminated glass slide, and a polymer brush (P(GMA-HEMA)) is grown through a surface-initiated atom transfer radical polymerization reaction (SI-ATRP). Upon the addition of MMPs, the FRET pairs are broken due to the specific cleavage of FRET-peptides by enzymes, resulting in the recovery of fluorescence signals and further enhancement by the MEF of GNRs. The fluorescence recovery degree provides a direct indicator for MMP activity. The PMMEFA exhibits excellent sensitivity, which enables to detect MMP-1, -2, -3, -7, -9, and -13 activities, with low limits of detection (LODs) of 1.7 fg mL−1, 0.3 fg mL−1, 2.0 fg mL−1, 1.8 fg mL−1, 2.2 fg mL−1 and 14.0 fg mL−1, respectively. To substantiate the practicability of PMMEFA, MMP activities were measured in a range of matrices, encompassing cell culture medium, serum, and tumor tissue homogenate, and MMP activities can be detected only in 0.15 μL serum and 0.025 mg tumor tissue.

Amyloid dye pairs as spectral sensors for enhanced detection and differentiation of misfolded proteins.

Protein misfolding with subsequent formation of cross-β-sheet-rich fibrils is a well-known pathological hallmark of various neurodegenerative conditions, including Alzheimer's disease (AD). Recent evidence suggests that specific protein conformations may be the primary drivers of disease progression, differentiation of which remains a challenge with conventional methods. We have previously described a unique phenomenon of light-induced fluorescence enhancement and spectral changes of the amyloid dyes K114 and BSB, and demonstrated its utility in characterizing different amyloid fibrils. In this study, we further characterize and explore the potential of photoconversion, coupled with dual-probe staining, for improved detection of heterogeneity of amyloids using silk fibers and 5xFAD mouse brain sections. BSB and K114 were paired with either Nile Red or MCAAD-3, aiming to increase the sensitivity and specificity of staining and misfolded protein detection via complementary binding and FRET. Principal component analysis of spectral data revealed significant differences between various amyloids, and was able to detect subtle amyloid pathology in the 5xFAD mouse background brain parenchyma.

A system of FRET dyes designed to assess the degree of nano-scale contact between surfaces for interfacial adhesion

HypothesisInterfacial adhesion caused by intermolecular forces only occur between surfaces at nano-scale contact (NSC), i.e., 0.1–0.4 nm and can be evaluated using Forster resonance energy transfer spectroscopy (FRET). For this, a suitable pair of fluorescent dyes must be selected, which spectroscopic properties will determine the FRET system performance. Here, we present a brand-new FRET dye system specifically designed to measure NSC in the distance range relevant for van-der-Waals and hydrogen bonding, i.e., below 1 nm. ExperimentsWe propose the FRET pair: 7-Amino-4-methyl-cumarin (C120) and 5(6)-Carboxy-2′,7′-dichlor-fluorescein (CDCF) with high quantum yield (QY, QYC120 = 0.91 and QYCDCF = 0.64) and a distance detection range of 0.6–2.2 nm (0.1 mM). Adhered-thin films with increasing NSC degrees are produced with ascending pressure from 1.5 to 150 bar. To validate the proposed FRET measurement, we are correlating the bonded films interfacial adhesion (separation energy) to the measured FRET intensity, indicating its degree of NSC. FindingsWe find that the proposed dyes are producing the desired FRET signal in adhered-thin films, for an interaction range of 0.6–2.2 nm, with high sensitivity due to the dye’s high quantum yields. The increasing adhesion in these films is only caused by its increase in NSC. We find that the adhesion strength, measured as the separation energy between the films, is correlated to the measured FRET signal. Hence, the introduced FRET system is accurately able to measure the degree of NSC between soft surfaces.

Real-time monitoring strategies for optimization of in vitro transcription and quality control of RNA.

RNA-based therapeutics and vaccines are opening up new avenues for modern medicine. To produce these useful RNA-based reagents, in vitro transcription (IVT) is an important reaction that primarily determines the yield and quality of the product. Therefore, IVT condition should be well optimized to achieve high yield and purity of transcribed RNAs. To this end, real-time monitoring of RNA production during IVT, which allows for fine tuning of the condition, would be required. Currently, light-up RNA aptamer and fluorescent dye pairs are considered as useful strategies to monitor IVT in real time. Fluorophore-labeled antisense probe-based methods can also be used for real-time IVT monitoring. In addition, a high-performance liquid chromatography (HPLC)-based method that can monitor IVT reagent consumption has been developed as a powerful tool to monitor IVT reaction in near real-time. This mini-review briefly introduces some strategies and examples for real-time IVT monitoring and discusses pros and cons of IVT monitoring methods.

FRET with MoS2 nanosheets integrated CRISPR/Cas12a sensors for robust and visual food-borne parasites detection

Recently, CRISPR/Cas associated biosensors have been shown to have great potential in sensing applications due to their high sensitivity and high base resolution. However, the signal reporter system containing two organic fluorescent dye pair is limited by high cost and less stability. In contrast, functional nanomaterials exhibit robust stability, excellent optical properties and low preparation cost, making them suitable reporters. In this study, a MoS2 nanosheets (NSs) improved CRISPR/Cas12a-based biosensing platform was constructed for the first time to detect food-borne parasites. MoS2 NSs were used as fluorescence quenchers and single-stranded DNA (ssDNA) discriminated carrier to construct CRISPR/Cas signal reporter system. The combination of recombinase polymerase amplification with MoS2 NSs modulated CRISPR/Cas12a helped achieve attomolar sensitivity for nucleic acid detection within 35 min. Moreover, the results were obtained using a portable apparatus, enabling visual detection at the point of care. The practical applicability of this biosensing platform was successfully achieved through the detection of anisakis in real samples. This study provides novel insights into exploring the feasibility of two-dimensional nanomaterials based reporter in the CRISPR/Cas12a system, as well as offers a reliable tool for on-site monitoring of parasites.

Highly chromophoric dual-terminus labeling of an intrinsically disordered native eukaryotic protein of interest at nanoscale

Chemical conjugation of purified proteins of interest (POIs) in Escherichia coli cells is effective for high expression but has limitations for highly chromogenic dual labeling of intrinsically disordered native proteins (IDNPs). Our probes can tag IDNPs using chemical conjugation during protein synthesis and folding while preserving biologically active structures in mammalian cells. We fluorescently labeled IDNPs in mammalian cells using pure fluorescent methionine and ATTO 565-biotin at the N-or C-terminus, respectively. The dual-labeled Tat protein was used as a model for IDNPs in HeLa cells and detected using Ni-NTA beads to estimate its highly chromogenic concentration. We also demonstrated highly chromogenic double labeling of genetically encoded fluorescent-Tat expression in eukaryotic cells using a single fluorescent dye pair with Förster resonance energy transfer (FRET) ratio and two-color correlation analysis. This study aims to solve native POI processing and achieve ultra-sensitive protein folding for biological and ecological applications at the nanoscale.

A comparative study of conventional FRET and light harvesting properties of Rh-110/Rh-6G and Rh-19/Rh-B organic dye pairs impregnated in sol-gel glasses

Two different pairs of laser dyes, Rhodamine-110 (Rh-110)/Rhodamine-6G (Rh-6G) and Rhodamine-19 (Rh-19)/Rhodamine-B (Rh-B) (the first dye in each pair as a donor and the second as an acceptor) were impregnated in silica samples prepared by the sol-gel method and spectroscopically studied using absorption and steady-state fluorescence techniques. The critical transfer distance (R0), actual distance (r) between the donor and acceptor, overlap integral [J()], FRET (fluorescence resonance energy transfer) efficiency (E), and antenna effect efficiency (AE) were investigated in detail based on the variation in acceptor concentration. The FRET efficiency, antenna effect efficiency, and actual donor-acceptor distance for Rh-110/Rh-6G and Rh-19/Rh-B dye pairs corresponding to acceptor concentration ranges (3.83 to 7.65) × 10−5 M l−1 and (3.71 to 8.34) × 10−5 M l−1, respectively, were found to be in the ranges of 57.38% to 74.89%, 36.97% to 24.13%, 5.44 nm to 4.77 nm, and 77.01%. Furthermore, maximum FRET efficiencies of 85.68% and 87.63% and antenna effect efficiencies of 36.97% and 40.95% for Rh-110/Rh-6G and Rh-19/Rh-B, respectively, were also reported. Our results demonstrate the superior FRET efficiency of Rh-19/Rh-B over Rh-110/Rh-6G dye pair in sol-gel glasses, while the antenna effect efficiency of Rh-110/Rh-6G is higher than that of Rh-19/Rh-B for the same donor to acceptor (D/A) ratio. Finally, Rh-110/Rh-6G is a better energy harvester than the Rh-19/Rh-B dye pair at the common D/A ratio. These results are explained in terms of molecular structure similarity, polarity, and rigidity of donor and acceptor.

A plasmonic fluorescent ratiometric temperature sensor for self-limiting hyperthermic applications utilizing FRET enhancement in the plasmonic field.

Nanoparticle mediated photo-induced hyperthermia holds much promise as a therapeutic solution for the management of diseases like cancer. The conventional methods of temperature measurements do not measure the actual temperature generated in the vicinity of the nanoparticles during illumination. In contrast, nano temperature sensors built on hyperthermic nanoparticles can relay local temperatures around the nanoparticles during thermal induction. In this study, we present a core shell construct consisting of a plasmonic core and a silica shell encapsulating a FRET pair of organic dyes for such application. The plasmonic core imparts photo-induced hyperthermic properties to the nanoconstruct, while the fluorescent shell enables ratiometric sensing of temperature. We see that even at a low dye encapsulation concentration, the shell displays a linear ratiometric fluorescence response to temperature and high energy transfer between the dye pair. Interestingly, Monte Carlo simulations, without considering the plasmonic core, show that the energy transfer in the system should be much smaller than that observed, confirming plasmon enhancement in the FRET energy transfer. We also show the ratiometric temperature measurement using these particles during photoinduced hyperthermia. This study suggests the use of plasmonic nanoparticles in the next generation "self-limiting" photothermal therapy.

Plasmon effect on triplet-singlet energy transfer in the dye-doped Langmuir-Blodgett films

The effect of silver plasmonic nanoparticles on triplet-singlet energy transfer in the donor-acceptor pair of organic dyes was studied. Layered dye films were prepared on the surface of glass and island silver films using the Langmuir-Blodgett method. Amphiphilic analogs of Rose bengal and polymethine dye were used as donor and acceptor of energy. A polymer monolayer was deposited between monolayers of donor and acceptor molecules to exclude the competing triplet-triplet energy transfer. The spectra of delayed fluorescence and phosphorescence of donor-acceptor films and the delayed luminescence lifetime of donor in these films have been measured. It is shown that a threefold increase in the fluorescence intensity and a twofold increase in the donor phosphorescence intensity are observed on silver island films. The successful triplet-singlet energy transfer is evidenced by both the quenching of donor centers and appearance of sensitized delayed fluorescence of the acceptor with the duration close to the donor triplet lifetime. In the presence of silver nanoparticles, in addition to enhancement in the intensity of the donor emission, an increase in the efficiency of tripletsinglet energy transfer was observed. The obtained results can be used in various optical devices.

In‐Silico Device Performance Prediction of Cosensitizer Dye Pairs for Dye‐Sensitized Solar Cells

AbstractEndeavors in the field of dye‐sensitized solar cells (DSCs) have shown great promise when adopting a data‐driven approach to materials discovery, such as successful molecular‐scale predictions of light‐harvesting chromophores. However, predictions of DSC dyes would become much more sophisticated if a molecular‐to‐macroscopic DSC device prediction methodology existed. Thereby, a fully computational pipeline is presented that predicts device‐performance parameters of DSCs which contain varying dye combinations. Optimal pairing of complementary dyes is identified via a data‐driven workflow that affords cosensitized DSCs with maximum power‐conversion efficiencies. Six high‐performing DSC dyes are paired with partner dyes that are screened from a database of 8488 compounds using sequential heuristic filters. Existing models that predict short‐circuit‐current density (JSC) and open‐circuit voltage (VOC) parameters are adapted to predict singly sensitized and cosensitized DSC performance. The predictions for Jsc values of singly sensitized devices match experimental literature values with comparable accuracy to more computationally costly methods. Five out of six dye pairings are predicted to have greater JSC values when cosensitized compared to their corresponding singly sensitized devices, including two pairs that show strong Jsc boosts of +13% and +12% when cosensitized. Thus, the prospect of an entirely in‐silico prediction pipeline for DSC performance that can be used to realize the fully automated design of optimized cosensitized DSCs is demonstrated.