Dye Binding Research Articles

Enhancing the optoelectronic properties of blended triphenylamine-betalain based dyes through tailoring the anchoring unit: a theoretical investigation

A series triphenyl-betalain organic dyes featuring carboxylic acid and nitro anchoring groups CH = C(X)COOH for the A1-X dyes and -CH = C(X)NO2 for the A2-X dyes, respectively, where X = CN, CH3, CCl3 and CF3 was evaluated for dye sensitised solar cells application. The geometrical structures, molecular orbitals and energies, light absorption patterns, free energies of electron injection and dye regeneration and binding to the semiconductor have been explored using DFT/TD-DFT methods. The nitro-based anchoring group resulted in pronounced red-shift in absorption spectra between 111 and 317 nm compared to carboxylic acid-based dyes. Attachment of the dyes to the semiconductor was modelled via binding to (TiO2)6H3 cluster; A2-X dyes exhibited more stable Dye@TiO2 complexes with binding energies (BEs) ranging between −4.08 and −2.88 eV compared to A1-X dyes with BEs range of −1.11 to −0.05 eV. The results evince that the dyes with CH = C(X)NO2 anchoring groups could be promising materials for light harvesting application.

Enzyme Fueled Dissipative Self-assembly of Guanine Functionalized Molecules and Their Cellular Behaviour.

Generally, an esterase lipase enzyme can hydrolyze specific substrates called esters in an aqueous solution. Herein, we investigate how a G-quadruplex self-assembly affects the hydrolysis equilibrium in reverse. The biocatalyst, lipase, activates the individual building-blocks through fuel consumption, causing them to undergo a higher degree of self-organization into nanofibers within spheres. We have synthesized five peptide-lipid-conjugated guanine base functionalized molecules to explore how the equilibrium can be shifted through reverse hydrolysis. Among these, NAC5 self-assembled into a G-quadruplex structure which has been confirmed by various spectroscopic techniques. The wide-angle powder XRD, ThT dye binding assay and circular dichroism study is carried out to support the presence of the G-quadruplex structure. The biocatalytic formation of nanofibers enclosed spheres is analyzed using CLSM, FE-SEM and HR-TEM experiments. Additionally, we assess the biocompatibility of the enzyme fueled dissipative self-assembled fibers enclosed spheres, as they have potential applications as a biomaterial in protocells. MTT assay is performed to check the cytotoxicity of G-quadruplex hydrogel, using HEK 293 and McCoy cell lines for viability assessment. Finally, the utility of the novel NAC5 hydrogel as a wound repairing biomaterial is demonstrated by cell migration experiment in a scratch assay.

Enhanced efficiency, electron lifetime, and eco-friendliness of dye-sensitized solar cells using nanofibrous TiO2/ZnO composite photoanodes and natural anthocyanin sensitizer

Semiconductor is the main part of the photoanode in dye-sensitized solar cells (DSSCs). In this study, an evaluation was conducted on two different morphological shapes of TiO2/ZnO semiconductors, namely nanoparticles (NPs) and nanofibers (NFs), to enhance the efficiency of DSSCs. The nanofibrous semiconductors were fabricated using the electrospinning technique. Furthermore, a natural sensitizer, anthocyanin dye, was used to promote the environmental friendliness of the fabricated cells. These cells were then compared to those that used a synthetic dye known as N719 to determine their efficiencies. The UV–vis results confirmed the extraction of anthocyanin from black plum fruits (Syzygium cumini). The structural characteristics of the composites were examined using XRD, FE-SEM, and BET experiments. The XRD results revealed that the anatase phase of TiO2 was dominant in the crystal structure of the semiconductors, while the crystallite sizes of the composites were 25.04 nm and 12.24 nm in NPs and NFs forms, respectively. The FE-SEM analysis revealed the formation of quasi-spherical NPs with an average diameter of 48.26 ± 17.75 nm and NFs with an average diameter of 153.99 ± 26.18 nm. The BET results showed that the surface characteristics of the nanocomposite were enhanced by changing the shape from NPs to NFs. Photovoltaic studies showed that utilizing NFs semiconductors in the photoanodes of DSSCs resulted in increased conversion efficiency of light to electricity. The results of the sun simulator studies confirmed that the use of natural dye led to a decrease in the effectiveness of the DSSCs. This phenomenon can be attributed to the weaker binding of the natural dye to the photoanodes. Furthermore, the EIS investigations illustrated that the electron lifetime in the natural dye is more than twice that of N719, leading to a reduced rate of electron recombination in the Syzygium cumini extract. The TiO2/ZnO NFs photoanodes exhibited superior performance compared to their nanoparticle counterparts in DSSCs. Their high surface area promoted superior dye adsorption and light absorption. Moreover, the interconnected nanofiber network facilitated efficient electron transport, minimizing recombination losses. Increasing the binding strength of the chosen natural dye to the photoanode will also make the cells more environmentally friendly and improve their ability to produce electricity.

Spectroscopic and solution properties characterization of quaternary ammonium ion containing polycations complexed with fluorescent rhodamine sulfonic acid dyes

Based on the growing range of applications for polycations in research and commercial materials, a continuing need exists to advance the fundamental knowledge and understanding of this class of materials. Spectroscopic and solution properties characterizations of noncovalently labeled, fluorescent Alexa Fluor® dye complexes of two commercial polycations, poly(2-(trimethylamino) ethyl methacrylate) monocation and poly[bis[2-chloroethyl] ether-alt-1,3-bis[3-(dimethylamino) propyl] urea] dication are reported to help address this need. A variety of fluorescence spectroscopic methods are used with a special emphasis on fluorescence correlation spectroscopy (FCS) which is applied to characterize the Stokes radius (RS) and equilibrium dissociation constants (Kd) of dye-polycation complexes at nanomolar dye concentrations. Resulting RS values indicate dye binding to individual polycation chains. Measured Kd values in the sub-micromolar range are consistent with strong dye binding. Increasing solution ionic strength with sodium chloride addition inhibits dye binding and decreases the RS of dye-polycation complexes due to size collapse of polycation chains. The complexes differ in their solution stability to ionic strength changes suggesting that both electrostatic and hydrophobic binding interactions influence dye binding. This study establishes the viability of noncovalent dye-polycation complexation in concert with FCS characterization as a general approach for investigating the properties of quaternary ammonium ion containing polycations in aqueous solution.

Investigation of Extracted Plasma Cell-Free DNA as a Biomarker in Foals with Sepsis.

Cell-free DNA (cfDNA) is fragmented extracellular DNA that is released from cells into various body fluids. Previously published data from adult horses supports cfDNA as a potential disease biomarker, but also shows that direct measurement in plasma is inaccurate due to matrix effect. It is currently unknown whether a similar matrix effect exists in foal plasma. Given this, the objectives of the current study were to investigate foal plasma for potential matrix effect during fluorescence measurement of cfDNA using a Qubit fluorometer, and to determine whether neat and/or extracted plasma cfDNA concentrations are significantly different in healthy, sick non-septic (SNS) or septic foals. We hypothesized that matrix effect would interfere with accurate fluorescent measurement of cfDNA in foal plasma. Further, we hypothesized that mean extracted cfDNA concentrations, and/or extracted cfDNA:neutrophil ratio, would be elevated in plasma of septic foals compared to healthy or SNS foals. Cell-free DNA was measured in neat plasma, and following DNA extraction with a commercial kit, from 60 foals. Foal plasma exhibited both autofluorescence and non-specific dye binding, confirming matrix effect. However, even with extraction, no significant difference was found in cfDNA concentrations, or cfDNA:neutrophil ratios, between healthy (sepsis score ≤ 5), SNS (sepsis score 6-11 and negative blood culture), or septic (sepsis score ≥ 12 ± positive blood culture) foals. Our data show that matrix effect interferes with accurate Qubit measurement of cfDNA in foal plasma and supports previous findings that plasma cfDNA concentrations are not associated with sepsis diagnosis in foals. Further research is needed to better understand neutrophil function and dysfunction in foal sepsis.

Effect of Mordants on Percentage Absorption of Rubia Cardifolia Dye

This study examines the impact of various mordants on the absorption of Indian Madder Root dye by cotton fabric. The Indian Madder Root dye, derived from the roots of the Indian madder plant, is a natural, eco-friendly alternative to synthetic dyes. The mordants used in this investigation include ferrous sulfate, copper sulfate, zinc sulfate, magnesium sulfate, aluminum potassium sulfate, and potassium dichromate. The absorption of the dye was measured using a colorimeter at a wavelength of 680 nm. The results show that the transition metal mordants, such as copper sulfate and ferrous sulfate, exhibit the highest percentage absorption, while magnesium sulfate shows the least absorption. The study highlights the significance of mordants in enhancing the binding of dyes to fabric, resulting in improved colorfastness and retention of color. The findings of this research have implications for the textile industry, particularly in the development of sustainable and environmentally friendly dyeing processes.

Efficient adsorption of cationic and anionic dyes using hydrochar nanoparticles prepared from orange peel

Hydrochar nanoparticles biosorbent (HCNPs), derived from orange peel was prepared via an in-situ hydrothermal carbonization method. The HCNPs were tested as an effective biosorbent for removing cationic MB (methylene blue) and anionic MO (methyl orange) dyes. The prepared HCNPs was characterized using FTIR, CHN/O, SEM-EDX, UV–visible, XRD, PZC-titration, BET, and XPS. The biosorbent exhibits a good specific surface-to-volume ratio (28.23 m2/g and 0.069 cc/g, respectively) and mesopores size (2.41 nm). UV–Vis data confirm the graphitic nature of the carbon structures. XPS analysis reveals a high enrichment of multiple oxygen functionalities (including C-OH, C = O, and –COO- groups) on the surface. The optimal initial pHs are 3.0 and 5.0–8.0 for removing MO and MB, respectively. Electrostatic attraction plays a substantial role, with other hydrophobic forces in dye removal and binding. Equilibrium is attained during 60/90 min, with half-biosorption-time (tHST) for MB and MO are 4.72 and 6.71 min, respectively. The highest biosorption capacities are 203.4 mg/g for MB and 113.3 mg/g for MO, closely correlated with physiochemical characteristics. Experimental data follows Langmuir isotherm and PSO kinetic models, indicating monolayer chemisorption on a homogeneous surface. The biosorption process for both dyes is exothermic and spontaneous. HCNPs show high durability over multiple biosorption/desorption cycles using HCl and NaOH (0.1 M) for MB and MO, respectively. These findings highlight the promising potential of HCNPs as efficient biosorbents for removing both anionic and cationic dyes.

Polymeric Microneedles Enhance Transdermal Delivery of Therapeutics.

This research presents the efficacy of polymeric microneedles in improving the transdermal permeation of methotrexate across human skin. These microneedles were fabricated from PLGA Expansorb® 50-2A and 50-8A and subjected to comprehensive characterization via scanning electron microscopy, Fourier-transform infrared spectroscopy, and mechanical analysis. We developed and assessed a methotrexate hydrogel for physicochemical and rheological properties. Dye binding, histological examinations, and assessments of skin integrity demonstrated the effective microporation of the skin by PLGA microneedles. We measured the dimensions of microchannels in the skin using scanning electron microscopy, pore uniformity analysis, and confocal microscopy. The skin permeation and disposition of methotrexate were researched in vitro. PLGA 50-8A microneedles appeared significantly longer, sharper, and more mechanically uniform than PLGA 50-2A needles. PLGA 50-8A needles generated substantially more microchannels, as well as deeper, larger, and more uniform channels in the skin than PLGA 50-2A needles. Microneedle insertion substantially reduced skin electrical resistance, accompanied by an elevation in transepidermal water loss values. PLGA 50-8A microneedle treatment provided a significantly higher cumulative delivery, flux, diffusion coefficient, permeability coefficient, and predicted steady-state plasma concentration; however, there was a shorter lag time than for PLGA 50-2A needles, base-treated, and untreated groups (p < 0.05). Conclusively, skin microporation using polymeric microneedles significantly improved the transdermal delivery of methotrexate.

Analytical Aspects of ANSA-BSA Association: A Thermodynamic and Conformational Approach.

Many studies have demonstrated the manner in which ANS interacts with bovine serum albumin (BSA), although they are limited by the extremely low solubility of dye. The present study demonstrates the binding of ANSA dye with BSA, and since this dye can easily replace ANS, it not only simplifies research but also improves sensor accuracy for serum albumin. A combination of calorimetry and spectroscopy has been employed to establish the thermodynamic signatures associated with the interaction of ANSA with the protein and the consequent conformational changes in the latter. The results of differential scanning calorimetry reveal that when the concentration of ANSA in solution is increased, the thermal stability of the protein increases substantially. The fluorescence data demonstrated a decrease in the binding affinity of ANSA with the protein when pH increased but was unable to identify a change in the mode of interaction of the ligand. ITC has demonstrated that the mode of interaction between ANSA and the protein varies from a single set of binding sites at pH 5 and 7.4 to a sequential binding site at pH 10, emphasizing the potential relevance of protein conformational changes. TCSPC experiments suggested a dynamic type in the presence of ANSA. Molecular docking studies suggest that ANSA molecules are able to find ionic centers in the hydrophobic pockets of BSA. The findings further imply that given its ease of use in experiments, ANSA may be a useful probe for tracking the presence of serum albumin and partially folded protein states.

Biomimetic nanoplatforms constructed from dialkylaminostyryl hetarene dyes and phospholipids exhibiting selective fluorescent response to specific proteins

The present work explores the specificity of supramolecular assemblies comprising dialkylaminostyrylhetarene dye molecules incorporated into phosphatidylcholine (PC) or phosphatidylserine (PS) aggregates. In PS-based assemblies, the dyes demonstrate a concentration-dependent fluorescent response, distinguishing anionic proteins such as bovine serum albumin (BSA) and pepsin from lysozyme (LYZ) in aqueous solutions. Conversely, no significant response is observed when the dyes are incorporated into the well-organized bilayers of neutral PC. The fluorescent response arises from the binding of dyes to proteins, leading to the detachment of dye molecules from the assemblies, rather than from the binding of proteins to the assemblies, although the latter process is facilitated by electrostatic attraction. Thus, both the poor ordering of PS molecules and the interfacial arrangement of the dyes are prerequisites for the fluorescent response of dye-PS aggregates. The structure of the dyes significantly impacts the spectral features of dye-PS and dye-protein assemblies. An optimal dye structure has been identified for the recognition of BSA, with a limit of detection (LOD) of 10.8 nM.

Non-cytotoxic 3-Acetylcoumarin as an Attractive Target for Human Monoamine Oxidase (HMAO) Enzymes.

Coumarins are a broad class of naturally occurring oxygen-heterocyclic compounds found in plants with diverse biological properties, making them attractive for evaluation as novel therapeutic agents. We herein report the in vitro cytotoxic and monoamine oxidase (MAO) inhibitory activities of 3-acetylcoumarins (6a-e). The cytotoxic activity was evaluated using crystal violet dye binding assay, and those compounds unable to induce cytotoxicity were further tested for the monoamine oxidase (MAO) activity using the MAO-GloTM kit. The 3-acetylcoumarins (6a-e) were non-cytotoxic (inactive) against MDA MB-231 (estrogen receptor-negative, ER-, highly invasive) and MCF-7 (estrogen receptor-positive, ER+, weakly invasive) breast cancer cell lines, but showed interesting MAOs inhibition activities. Among the synthesized compounds, 3-acetylcoumarin bearing dichloro (-diCl) (6d; IC50=0.31±0.04 μM) at Carbon-7, 8 positions showed higher inhibition, MAO B/A non-selectivity (selectivity index, SI=3.10), reversible inhibition against the hMAO-B enzyme, and neuroprotection against H2O2-treated human neuroblastoma (N2a) cells. Compound (6d) can be considered a promising scaffold for further investigation in developing hMAO-B inhibitors (MAOIs).

Chemical reaction driven self-assembly of a nucleobase functionalized molecule

Molecular self-assembly is an important aspect in the nano technology and nano science which is exploited to produce complex supramolecular nanoarchitectures. Different strategies are used to achieve the molecular self-assembly in the laboratory. Here, the methylation reaction is used to induce the supramolecular order in the synthesized guanine functionalized molecules. Methylation is an important biological process which is used for DNA methylation. The methylation reaction driven formation of higher order self-assembly is discussed. The formation of the activated building blocks is studied using high-performance liquid chromatography. The N7 methylation is observed majorly. The pH of the medium is decreased with the progress of the reaction. The progress of self-assembly upon the addition of dimethyl sulphate is monitored by fluorescence, circular dichroism spectroscopy, 1H nuclear magnetic resonance and turbidity experiments. Additionally, Thioflavin T dye binding, wide-angle powder X-ray diffraction, circular dichroism spectroscopy experiments are performed to establish the formation of the chemical reaction driven self-assembled hydrogel. The formation of the nanofiber inside the hydrogel is confirmed by performing various microscopic experiments.

One-pot growth and characterization of α-Fe2O3/β-FeOOH micro-nanosystem for industrial dye removal

A micro-nanosystem of α-Fe2O3/β-FeOOH was developed for the first time using a one-pot hydrothermal method. The synthesis mechanism of the simultaneous development of micro-sized α-Fe2O3 and nano-sized β-FeOOH is discussed. The structural properties studied via X-ray diffraction (XRD) revealed the presence of both phases, whereas the FESEM study confirms that α-Fe2O3 and β-FeOOH are of microdisc and nanohusk type, respectively. Considering the two different shapes and sizes of particles in a micro-nanosystem, α-Fe2O3/β-FeOOH was used to remove rhodamine B dye. The hydrogen bonding between the adsorbent and the RhB dye influences the binding properties. The mixed-shaped iron-based particles have provided maximum adsorbing sites which have strengthened the bonding between the adsorbent and adsorbate. The FTIR study was used to confirm the binding of RhB dye with the micro-nanosystems. Adsorption capacity and removal efficiency for α-Fe2O3/β-FeOOH micro-nanosystem adsorbent were found to be 727 mg g−1 and 91 % respectively. The binding mechanism along with the adsorption isotherm and adsorption kinetic study, has been discussed. The adsorption isotherm study confirms that the adsorption process followed the Freundlich isotherm model. The adsorption kinetic study suggested the well-fitting of pseudo-first-order kinetics and the Elovich model with the adsorption process. The reusability study of the micro-nanosystem confirms that the micro-nano system can retain the highest removal efficiency up to 4th cycle. Further, the fabricated micro-nanosystem was applied for the removal of RhB dye from the real water samples, collected from different sources.

Development of D-π-A organic dyes for discriminating HSA from BSA and study on dye-HSA interaction

HSA (human serum albumin), a most abundant protein in blood serum, plays a key role in maintaining human health. Abnormal HSA level is correlated with many diseases, and thus has been used as an essential biomarker for therapeutic monitoring and biomedical diagnosis. Development of small-molecule fluorescent probes allowing the selective and sensitive recognition of HSA in in vitro and in vivo is of fundamental importance in basic biological research as well as medical diagnosis. Herein, we reported a series of new synthesized fluorescent dyes containing D-π-A constitution, which exhibited different optical properties in solution and solid state. Among them, dye M−H−SO3 with a hydrophilic sulfonate group at electron-acceptor part displayed selectivity for discrimination of HSA from BSA and other enzymes. Upon binding of dye M−H−SO3 with HSA, a significant fluorescence enhancement with a turn-on ratio about 96-fold was triggered. The detection limit was estimated to be ∼ 40 nM. Studies on the interaction mechanism revealed that dye M−H−SO3 could bind to site III of HSA with a 1:1 binding stoichiometry. Furthermore, dye M−H−SO3 has been applied to determine HSA in real urine samples with good recoveries, which provided a useful method for HSA analysis in biological fluids.

Nucleotide-induced ClpC oligomerization and its non-preferential association with ClpP isoforms of pathogenic Leptospira

Bacterial caseinolytic protease-chaperone complexes participate in the elimination of misfolded and aggregated protein substrates. The spirochete Leptospira interrogans possess a set of Clp-chaperones (ClpX, ClpA, and ClpC), which may associate functionally with two different isoforms of LinClpP (ClpP1 and ClpP2). The L. interrogans ClpC (LinClpC) belongs to class-I chaperone with two active ATPase domains separated by a middle domain. Using the size exclusion chromatography, ANS dye binding, and dynamic light scattering analysis, the LinClpC is suggested to undergo nucleotide-induced oligomerization. LinClpC associates with either pure LinClpP1 or LinClpP2 isoforms non-preferentially and with equal affinity. Regardless, pure LinClpP isoforms cannot constitute an active protease complex with LinClpC. Interestingly, the heterocomplex LinClpP1P2 in association with LinClpC forms a functional proteolytic machinery and degrade β-casein or FITC-casein in an energy-independent manner. Adding either ATP or ATPγS further fosters the LinClpCP1P2 complex protease activity by nurturing the functional oligomerization of LinClpC. The antibiotic, acyldepsipeptides (ADEP1) display a higher activatory role on LinClpP1P2 protease activity than LinClpC. Altogether, this work illustrates an in-depth study of hetero-tetradecamer LinClpP1P2 association with its cognate ATPase and unveils a new insight into the structural reorganization of LinClpP1P2 in the presence of chaperone, LinClpC to gain protease activity.

Comparative evaluation of physical and chemical enhancement techniques for transdermal delivery of linagliptin

Linagliptin is a dipeptidyl peptidase-4 inhibitor used for the management of type-2 diabetes. US FDA-approved products are available exclusively as oral tablets. The inherent drawbacks of the oral administration route necessitate exploring delivery strategies via other routes. In this study, we investigated the feasibility of transdermal administration of linagliptin through various approaches. We compared chemical penetration enhancers (oleic acid, oleyl alcohol, and isopropyl myristate) and physical enhancement techniques (iontophoresis, sonophoresis, microneedles, laser, and microdermabrasion) to understand their potential to improve transdermal delivery of linagliptin. To our knowledge, this is the first reported comparison of chemical and physical enhancement techniques for the transdermal delivery of a moderately lipophilic molecule. All physical enhancement techniques caused a significant reduction in the transepithelial electrical resistance of the skin samples. Disruption of the skin’s structure post-treatment with physical enhancement techniques was further confirmed using characterization techniques such as dye binding, histology, and confocal microscopy. In vitro permeation testing (IVPT) demonstrated that the passive delivery of linagliptin across the skin was < 5 µg/sq.cm. Two penetration enhancers - oleic acid (93.39 ± 8.34 µg/sq.cm.) and oleyl alcohol (424.73 ± 42.86 µg/sq.cm.), and three physical techniques - iontophoresis (53.05 ± 0.79 µg/sq.cm.), sonophoresis (141.13 ± 34.22 µg/sq.cm.), and laser (555.11 ± 78.97 µg/sq.cm.) exceeded the desired target delivery for therapeutic effect. This study established that linagliptin is an excellent candidate for transdermal delivery and thoroughly compared chemical penetration and physical transdermal delivery strategies.

The Occurrence, Antibiotic Susceptibility Pattern and Multiple Drug Resistance Index Studies of E. coli from Fresh Meats in Marathwada Region of Maharashtra

Background: Foodborne diseases are a huge social burden in both developed and underdeveloped countries. Escherichia coli is a natural inhabitant of the human and warm-blooded animal digestive tracts. It is considered as an indicator organism for direct or indirect faecal contamination of raw meats. The presence of antibiotic-resistant microorganisms in meat accounts for a public health risk. Methods: In current study, all 39 characterized E. coli isolates recovered on screening 405 fresh meat samples collected from different butcher shops in the Udgir city, Marathwada region of Maharashtra were also tested for Congo red dye binding assay and haemolysis assay. Antimicrobial resistance profiling of recovered isolates was carried out using the disc diffusion method. Result: Overall occurrence of E. coli in fresh meats was noted to the tune of 9.63% and the corresponding group wise occurrence in chicken and chevon samples were 12% and 7.32%, respectively. All E. coli tested positive in the Congo red dye binding assay and haemolysis assay, suggesting virulent nature of isolated organisms. On screening for antimicrobial resistance, these isolates were found resistant to mainly â-lactams (94.87%), lincosamides (84.62%), 4th Generation cephalosporins (82.05% each), DHFR Inhibitors (64.10%), glycopeptides (51.28%). The multidrug resistant isolates showed resistance to a minimum of 4 and maximum of 12 antibiotics with MAR index ranging from 0.27 to 0.8 and 9 resistance patterns.

Prediction of the Feasibility of Using the ≪Gold Standard≫ Thioflavin T to Detect Amyloid Fibril in Acidic Media.

Ordered protein aggregates, amyloid fibrils, form toxic plaques in the human body in amyloidosis and neurodegenerative diseases and provide adaptive benefits to pathogens and to reduce the nutritional value of legumes. To identify the amyloidogenic properties of proteins and study the processes of amyloid fibril formation and degradation, the cationic dye thioflavin T (ThT) is the most commonly used. However, its use in acidic environments that induce amyloid formation in vitro can sometimes lead to misinterpretation of experimental results due to electrostatic repulsion. In this work, we show that calculating the net charge per residue of amyloidogenic proteins or peptides is a simple and effective approach for predicting whether their fibrils will interact with ThT at acidic pH. In particular, it was shown that at pH 2, proteins and peptides with a net charge per residue > +0.18 are virtually unstained by this fluorescent probe. The applicability of the proposed approach was demonstrated by predicting and experimentally confirming the absence of ThT interaction with amyloids formed from green fluorescent (sfGFP) and odorant-binding (bOBP) proteins, whose fibrillogenesis was first carried out in an acidic environment. Correct experimental evidence that the inability to detect these fibrils under acidic conditions is precisely because of the lack of dye binding to amyloids (and not their specific structure or the low fluorescence quantum yield of the bound dye) and that the number of ThT molecules associated with fibrils increases with decreasing acidity of the medium was obtained by using the equilibrium microdialysis approach.

The use of fluorescence lifetime imaging (FLIM) for in situ microbial detection in complex mineral substrates.

The utility of fluorescence lifetime imaging microscopy (FLIM) for identifying bacteria in complex mineral matrices was investigated. Baseline signals from unlabelled Bacillus subtilis and Euglena gracilis, and Bacillus subtilis labelled with SYTO 9 were obtained using two-photon excitation at 730, 750 and 800nm, identifying characteristic lifetimes of photosynthetic pigments, unpigmented cellular autofluorescence, and SYTO 9. Labelled and unlabelled B. subtilis were seeded onto marble and gypsum samples containing endolithic photosynthetic cyanobacteria and the ability to distinguish cells from mineral autofluorescence and nonspecific dye staining was examined in parallel with ordinary multichannel confocal imaging. It was found that FLIM enabled discrimination of SYTO 9 labelled cells from background, but that the lifetime of SYTO 9 was shorter in cells on minerals than in pure culture under our conditions. Photosynthetic microorganisms were easily observed using both FLIM and confocal. Unlabelled, nonpigmented bacteria showed weak signals that were difficult to distinguish from background when minerals were present, though cellular autofluorescence consistent with NAD(P)H could be seen in pure cultures, and phasor analysis permitted detection on rocks. Gypsum and marble samples showed similar autofluorescence profiles, with little autofluorescence in the yellow-to-red range. Lifetime or time-gated imaging may prove a useful tool for environmental microbiology. LAY DESCRIPTION: The standard method of bacterial enumeration is to label the cells with a fluorescent dye and count them under high-power fluorescence microscopy. However, this can be difficult when the cells are embedded in soil and rock due to fluorescence from the surrounding minerals and dye binding to ambiguous features of the substrate. The use of fluorescence lifetime imaging (FLIM) can disambiguate these signals and allow for improved detection of bacteria in environmental samples.

NIR-II Protein-Escaping Dyes Enable High-Contrast and Long-Term Prognosis Evaluation of Flap Transplantation.

Real-time vascular positioning, postoperative flap monitoring, and vascular reconstruction assessment are of great importance in flap transplantation. Cyanine dyes offer the advantage of high resolution in the Near-infrared-II (NIR-II)imaging window. However, the nonspecific binding of many cyanine dyes to endogenous albumin leads to high organ accumulation and skin absorption, resulting in low-quality imaging and poor reproducibility of contrast during long-term (e.g., 7 days) postoperative monitoring. Here, a novel strategy is proposed that can be widely applied to prevent protein binding for NIR-I/IICl-containing cyanine dyes. This strategy produces protein-escaping dyes, ensuring high fluorescence enhancement in the blood with rapid clearance and no residual fluorescence, allowing for short-term repeatable injections for vascular imaging. This strategy in the perioperative monitoring of pedicle perforator flap models in mice and rats is successfully applied. Furthermore, leveraging the universality of this strategy, multiple nonoverlapping protein-escaping probes that achieve dual-excitation (808 and 1064nm) interference-free imaging of nerve-vessel and tumor-vessel simultaneously are designed and synthesized. These protein-escaping dyes enable long-term repeatable dual-color imaging of tumor localization, resection, and tumor-vessel reconstruction at the wound site.