Diagnosis and Vulnerability Risk Assessment of Atherosclerotic Plaques Using an Amino Acid-Assembled Near-Infrared Ratiometric Nanoprobe.

To reduce the risk of atherosclerotic disease, it is necessary to not only diagnose the presence of atherosclerotic plaques but also assess the vulnerability risk of plaques. Accurate detection of the reactive oxygen species (ROS) level at plaque sites represents a reliable way to assess the plaque vulnerability. Herein, through a simple one-pot reaction, two near-infrared (NIR) fluorescent dyes, one is ROS responsive and the other is inert to ROS, are coassembled in an amphiphilic amino acid-assembled nanoparticle. In the prepared NIR fluorescent amino acid nanoparticle (named FANP), the fluorescent properties and ROS-responsive behaviors of the two fluorescent dyes are well maintained. Surface camouflage through red blood cell membrane (RBCM) encapsulation endows the finally obtained FANP@RBCM nanoprobe with not only further reduced cytotoxicity and improved biocompatibility but also increased immune escape capability, prolonged blood circulation time, and thus enhanced accumulation at atherosclerotic plaque sites. In vitro and in vivo experiments demonstrate that FANP@RBCM not only works well in probing the occurrence of atherosclerotic plaques but also enables plaque vulnerability assessment through the accurate detection of the ROS level at plaque sites in a reliable ratiometric mode, thereby holding great promise as a versatile tool for the diagnosis and risk assessment of atherosclerotic disease.

Fluorescence and Biochemical Assessment of the Chitin and Chitosan Content of Cryptococcus.

The cell wall of the fungal pathogens Cryptococcus neoformans and C. gattii is critical for cell wall integrity and signaling external threats to the cell, allowing it to adapt and grow in a variety of changing environments. Chitin is a polysaccharide found in the cell walls of fungi that is considered to be essential for fungal survival. Chitosan is a polysaccharide derived from chitin via deacetylation that is also essential for cryptococcal cell wall integrity, fungal pathogenicity, and virulence. Cryptococcus has evolved mechanisms to regulate the amount of chitin and chitosan during growth under laboratory conditions or during mammalian infection. Therefore, levels of chitin and chitosan have been useful phenotypes to define mutant Cryptococcus strains. As a result, we have developed and/or refined various qualitative and quantitative methods for measuring chitin and chitosan. These techniques include those that use fluorescent probes that are known to bind to chitin (e.g., calcofluor white and wheat germ agglutinin), as well as those that preferentially bind to chitosan (e.g., eosin Y and cibacron brilliant red 3B-A). Techniques that enhance the localization and quantification of chitin and chitosan in the cell wall include (i) fluorescence microscopy, (ii) flow cytometry, (iii) and spectrofluorometry. We have also modified two highly selective biochemical methods to measure cellular chitin and chitosan content: the Morgan-Elson and the 3-methyl-2-benzothiazolone hydrazine hydrochloride (MBTH) assays, respectively.

A novel ESIPT fluorescent probe for early detection and assessment of ferroptosis-mediated acute kidney injury via peroxynitrite fluctuation.

BACKGROUND: Acute kidney injury (AKI) poses a severe risk to public health, mostly manifested by damage and death of renal tubular epithelial cells. However, routine blood examination, a conventional approach for clinical detection of AKI, is not available for identifying early-stage AKI. Plenty of reported methods were lack of early biomarkers and real time evaluation tools, which resulted in a vital challenge for early diagnosis of AKI. Therefore, developing novel probes for early detection and assessment of AKI is exceedingly crucial. RESULTS: Based on ESIPT mechanism, a new fluorescent probe (MEO-NO) with 2-(2'-hydroxyphenyl) benzothiazole (HBT) derivatives as fluorophore has been synthesized for dynamic imaging peroxynitrite (ONOO-) levels in ferroptosis-mediated AKI. Upon the addition of ONOO-, MEO-NO exhibited obvious fluorescence changes, a significant Stokes shift (130 nm) and rapid response (approximately 45 s), and featured exceptional sensitivity (LOD = 7.28 nM) as well as high selectivity from the competitive species at physiological pH. In addition, MEO-NO was conducive to the biological depth imaging ONOO- in cells, zebrafish, and mice. Importantly, MEO-NO could monitor ONOO- levels during sorafenib-induced ferroptosis and CP-induced AKI. With the assistance of MEO-NO, we successfully visualized and tracked ONOO- variations for early detection and assessment of ferroptosis-mediated AKI in cells, zebrafish and mice models. SIGNIFICANCE AND NOVELTY: Benefiting from the superior performance of MEO-NO, experimental results further demonstrated that the levels of ONOO- was overexpressed during ferroptosis-mediated AKI in cells, zebrafish, and mice models. The developed novel probe MEO-NO provided a strong visualization tool for imagining ONOO-, which might be a potential method for the prevention, diagnosis, and treatment of ferroptosis-mediated AKI.

Assessment of Mitochondrial Oxygen Consumption Using a Plate Reader-based Fluorescent Assay.

Mitochondria serve many important functions, including cellular respiration, ATP production, controlling apoptosis, and acting as a central hub of metabolic pathways. Therefore, experimentally assessing mitochondrial functionality can provide insight into variations among different populations or disease states. Additionally, it is valuable to assess whether isolated mitochondria are healthy enough to proceed with experiments. One characteristic often used to compare mitochondrial function in different samples is the rate of oxygen consumption. Oxygen consumption and subsequent calculation of the respiratory control ratio in either intact cells or mitochondria isolated from tissue can serve all three purposes. Using mitochondria isolated from the livers of brush lizards in conjunction with a phosphorescent probe that is sensitive to the fluctuations in oxygen concentration of a solution, we measured oxygen consumption using a fluorescent plate reader. This method is not only quick and efficient but also can be conducted with a small amount of mitochondria and without the need for specialized equipment. The step-by-step protocol described here increases the accessibility of mitochondrial functional assessment to researchers.

Development of a rhodamine-based fluorescent probe for ATP detection for potential applications in meat freshness assessment.

Adenosine triphosphate (ATP) plays a vital role in physiological processes and is an essential indicator of microbial content in food. Herein, a new sensitive, rapid and water-soluble probe for ATP detection was developed. Rhodamine B and pentaethylenehexamine were employed to design and synthesise the probe rhodamine-pentaethylenehexamine (RP) for selective ATP detection. The synthesised probe RP was characterized using Fourier transform infrared, NMR and dynamic light scattering size distributions. Upon the addition of ATP, the probe exhibited a distinct change in fluorescence intensity, with fluorescence emission at 580 nm. A linear relationship was observed between fluorescence intensity and ATP concentrations at 0-50 µmol/L, with a limit of detection of 10.97 × 10-9 mol/L. The results of the zeta potential and molecular dynamics simulation demonstrated that the detection mechanism of the probe RP is associated with the electrostatic adsorption interaction between the multi-positively charged sites of RP and the negatively charged triphosphate structure of ATP. Our study provides new insights into improving charge site identification in small molecule detection. Furthermore, the successful detection of ATP on meat surfaces indicates that RP has the potential to assess meat freshness.

Multiparameter Assessment of Foam Cell Formation Progression Using a Dual-Color Switchable Fluorescence Probe.

The assessment of atherosclerosis (AS) progression has emerged as a prominent area of research. Monitoring various pathological features of foam cell (FC) formation is imperative to comprehensively assess AS progression. Herein, a simple benzospiropyran-julolidine-based probe, BSJD, with switchable dual-color imaging ability was developed. This probe can dynamically and reversibly adjust its molecular structure and fluorescent properties in different polar and pH environments. Such a polarity and pH dual-responsive characteristic makes it superior to single-responsive probes in dual-color imaging of lipid droplets (LDs) and lysosomes as well as monitoring their interaction. By simultaneously tracking various pathological features, including LD accumulation and size changes, lysosome dysfunction, and dynamically regulated lipophagy, more comprehensive information can be obtained for multiparameter assessment of FC formation progression. Using BSJD, not only the activation of lipophagy in the early stages and inhibition in the later phases during FC formation are clearly observed but also the important roles of lipophagy in regulating lipid metabolism and alleviating FC formation are demonstrated. Furthermore, BSJD is demonstrated to be capable of rapidly imaging FC plaque sites in AS mice with fast pharmacokinetics. Altogether, BSJD holds great promise as a dual-color organelle-imaging tool for investigating disease-related LD and lysosome changes and their interactions.

Comprehensive assessment of 12 commercial DNA-binding dyes as alternatives to ethidium bromide for agarose gel electrophoresis.

Staining and visualization of the nucleic acid bands on agarose gels using ethidium bromide (EB) has been a widely used technique in molecular biology. Although it is an efficient dye for this purpose, EB is known to be mutagenic and genotoxic in humans. This led to the emergence of various alternative dyes, which were claimed to be safer and more efficient than EB. However, these dyes portray varied sensitivity and interference with the electrophoretic mobility of nucleic acids. This work aimed at assessing ten nucleic acid-binding dyes and two prestained dyes for these properties by three staining techniques, such as precasting, preloading, and poststaining. Of these, preloading was not suitable for any of the dye while poststaining worked optimal for most of them. Precasting was suitable for only four dyes viz. DNA Stain G, SYBR™ safe, EZ-Vision® in-gel, and LabSafe™. Poststaining was, in general, a costlier method than precasting. The work gives a comprehensive understanding of the performance of nucleic acid-binding dyes for routine molecular biology experiments.

Tailored Zwitterionic Hemicyanine Reporters for Early Diagnosis and Prognostic Assessment of Acute Renal Failure.

Drug-induced renal failure (DIRF) poses a serious medical complication with high mortality risk. However, early diagnosis or prognosis of DIRF remain challenging, as current methods rely on detecting late-stage biomarkers. Herein we present a library of zwitterionic unimolecular hemicyanines (ZCs) available for constructing activatable reporters to detect DIRF since its initial stage. Zwitterionic properties of these probes are achieved through interspersedly integrating alkyl sulfonates and quaternary ammonium cations onto hemicyanine skeleton, which result in record low plasma protein binding (<5 %) and remarkable renal clearance efficiencies (≈96 %). An activatable reporter ZCRR is further developed by masking the optimal candidate ZC6 with a tetrapeptide specifically cleavable by caspase-8, an initiating indicator of apoptosis. In living mice with cisplatin-induced DIRF, systematically administered ZCRR efficiently accumulates in kidneys and responds to elevated caspase-8 for near-infrared fluorescence signals 'turn-on', enabling sensitive detection of intrarenal apoptosis 60 h earlier than clinical methods, and precise evaluation of apoptosis remediation effects by different medications on DIRF mice. As it's urinary excretable, ZCRR also allows for remote detection of DIRF and predicting renoprotective efficacy through in vitro optical urinalysis. This study thus presents unimolecular renal clearable scaffolds that are applicable to developing versatile activatable reporters for renal diseases management.

Rational Design of Cost-Effective 4-Styrylcoumarin Fluorescent Derivatives for Biomolecule Labeling.

Fluorescent labels are key tools in a wide range of modern scientific applications, such as fluorescence microscopy, flow cytometry, histochemistry, direct and indirect immunochemistry, and fluorescence in situ hybridization (FISH). Small fluorescent labels have important practical advantages as they allow maximizing the fluorescence signal by binding multiple fluorophores to a single biomolecule. At present, the most widely used fluorescent labels available present small Stokes shifts and are too costly to be used in routine applications. In this work we present four new coumarin derivatives, as promising and inexpensive fluorescent labels for biomolecules, obtained through a cost-effective, efficient, and straightforward synthetic strategy. Density functional theory and time-dependent density functional theory calculations of the electronic ground and lowest-lying singlet excited states were carried out in order to gain insights into the observed photophysical properties.

Quantitative assessment of lipophilic membrane dye-based labelling of extracellular vesicles by nano-flow cytometry.

Although lipophilic membrane dyes (LMDs) or probes (LMPs) are widely used to label extracellular vesicles (EVs) for detection and purification, their labelling performance has not been systematically characterized. Through concurrent side scattering and fluorescence detection of single EVs as small as 40 nm in diameter by a laboratory-built nano-flow cytometer (nFCM), present study identified that (1) PKH67 and PKH26 could maximally label ∼60%-80% of EVs isolated from the conditioned cell culture medium (purity of ∼88%) and ∼40%-70% of PFP-EVs (purity of ∼73%); (2) excessive PKH26 could cause damage to the EV structure; (3) di-8-ANEPPS and high concentration of DiI could achieve efficient and uniform labelling of EVs with nearly 100% labelling efficiency for di-8-ANEPPS and 70%-100% for DiI; (4) all the four tested LMDs can aggregate and form micelles that exhibit comparable side scatter and fluorescence intensity with those of labelled EVs and thus hardly be differentiate from each other; (5) as the LMD concentration went up, the particle number of self-aggregates increased while the fluorescence intensity of aggregates remained constant; (6) PKH67 and PKH26 tend to form more aggregated micelles than di-8-ANEPPS and DiI, and the effect of LMD self-aggregation can be negligible at optimal staining conditions. (7) All the four tested LMDs can label almost all the very-low-density lipoprotein (VLDL) particles, indicating potential confounding factor in plasma-EV labelling. Besides, it was discovered that DSPE-PEG2000 -biotin can only label ∼50% of plasma-EVs. The number of LMP inserted into the membrane of single EVs was measured for the first time and it was confirmed that membrane labelling by lipophilic dyes did not interfere with the immunophenotyping of EVs. nFCM provides a unique perspective for a better understanding of EV labelling by LMD/LMP.

Fabrication of a paper-based facile and low-cost microfluidic device and digital imaging technique for point-of-need monitoring of hypochlorite.

Lab-on-a-paper-based devices are promising alternatives to the existing arduous techniques for point-of-need monitoring. The present work reports an instant and facile method to produce a microfluidic paper-based analytical device (µPAD). The fabricated µPAD has been used to detect hypochlorite (OCl-) by incorporating newly synthesized chromo-fluorogenic ratiometric probes 1 and 2 into the sample reception zone. The probes showed high selectivity and fast response (<10 s) toward OCl- with an excellent linear relationship in the concentration range of 0-100 µM. The concentration-dependent fluorometric change driven by the reaction of 1@µPAD with OCl- has been monitored using gel-doc imaging systems, which is unprecedented. Digitizing the intensity of the colour solution with different mathematical models of colour has developed a straightforward method for monitoring OCl- without any interference from its competitors. 1@µPAD can detect OCl- at ∼10 times lower than the WHO recommended limit. The detection limit of 1@µPAD via a digital camera-based fluorescence technique was found to be better over digital camera-based cuvette assays. Therefore, 1@µPAD has been successfully utilized to monitor OCl- in actual environmental water samples with portability, ease of use, and sensitivity. The analytical RSD was found to be ≤3% based on fluorimetric detection using 1@µPAD. The chemodosimetric reaction between OCl- and the probe was evidenced by UV-vis and fluorescence spectroscopy, 1H NMR, and ESI-MS. The rapid response time, biocompatibility, low cytotoxicity, 100% aqueous solubility, ratiometric feature, and exclusive OCl- selectivity over other competitive ROS/RNS successfully lead to the application of the probes for bioimaging of exogenous as well as endogenous OCl- in normal cells (HEK293) and cancerous cells (HeLa).

Detection of exposed phosgene in household bleach: development of a selective and cost-effective sensing tool.

Sensing of gaseous environment pollutants and health hazards is in demand these days and in this regard, lethal phosgene has emerged as a leading entrant. In this contribution, we have successfully developed a facile chemodosimeter (ANO) based on an anthracene fluorophore and oxime recognition site with an interesting mechanism to sense lethal phosgene evolved from bleaching powder, a very popular disinfectant and sanitizer. The ANO probe is highly competent in recognizing deadly phosgene in solution and in the gaseous phase with a detection limit in the nanomolar range (1.52 nM). The sensing mechanism is confirmed by UV-vis, emission spectroscopy, mass spectrometry, and computational studies.

A wearable gloved sensor based on fluorescent Ag nanoparticles and europium complexes for visualized assessment of tetracycline in food samples.

The abuse of tetracycline antibiotics leads to accumulating residues in the human body, seriously affecting human health. Establishing a sensitive, efficient, and reliable method for qualitative and quantitative detection of tetracycline (TC) is necessary. This study integrated silver nanoclusters and europium-based materials into the same nano-detection system to construct a visual and rapid TC sensor with rich fluorescence color changes. The nanosensor has the advantages of a low detection limit (10.5 nM), high detection sensitivity, fast response, and wide linear range (0-30 µM), which can meet the analysis requirements of different types of food samples. In addition, portable devices based on paper and gloves were designed. Through the smartphone's chromaticity acquisition and calculation analysis application (APP), the real-time rapid visual intelligent analysis of TC in the sample can be realized, which guides the intelligent application of multicolor fluorescent nanosensors.

Activated Two-Photon Near-Infrared Ratiometric Fluorescent Nanoprobe for ONOO- Detection and Early Diagnosis and Assessment of Liver Injury.

Liver injury poses a serious threat to human health and growing evidence suggests that it is closely associated with a biomarker (peroxynitrite, ONOO-). Therefore, considering that the relationship of ONOO- levels with the occurrence and development of liver injury disease remains a challenge, an urgent need exists to develop a reliable and robust tool for its visual rapid diagnosis and assessment. Herein, a two-photon near-infrared (TP-NIR) ratiometric fluorescent nanoprobe (NTC) based on a fluorescence resonance energy transfer (FRET) strategy was designed, synthesized, and characterized, which had the advantages of good water solubility, low background interference, deep tissue penetration, and high imaging resolution. Specially, NTC was constructed by self-assembly of an alkynyl group of a small-molecule fluorescent probe (NR) via click chemistry grafting onto azide chitosan (natural polymeric nanomaterial). NR contained acceptor 1 (NIR fluorophore) and donor 3 (D-π-A structure of naphthalimide derivative fluorophore) with outstanding TP properties that could be activated by ONOO- for the ratiometric detection of ONOO-. Furthermore, in the presence of ONOO-, NTC exhibited a short response time (∼10 s) and high selectivity and sensitivity toward ONOO- with an excellent detection limit as low as 15.3 nM over other reactive oxygen/nitrogen species. Notably, NTC has been successfully employed for ONOO- detection and imaging in living HepG2 cells, liver injury mice tissues, and mice models with satisfactory results. Thus, the construction of this NTC nanoprobe can provide a robust molecule tool for enabling early diagnosis and assessment of liver injury in the future.

Dissecting the mechanisms of environment sensitivity of smart probes for quantitative assessment of membrane properties.

The plasma membrane, as a highly complex cell organelle, serves as a crucial platform for a multitude of cellular processes. Its collective biophysical properties are largely determined by the structural diversity of the different lipid species it accommodates. Therefore, a detailed investigation of biophysical properties of the plasma membrane is of utmost importance for a comprehensive understanding of biological processes occurring therein. During the past two decades, several environment-sensitive probes have been developed and become popular tools to investigate membrane properties. Although these probes are assumed to report on membrane order in similar ways, their individual mechanisms remain to be elucidated. In this study, using model membrane systems, we characterized the probes Pro12A, NR12S and NR12A in depth and examined their sensitivity to parameters with potential biological implications, such as the degree of lipid saturation, double bond position and configuration (cis versus trans), phospholipid headgroup and cholesterol content. Applying spectral imaging together with atomistic molecular dynamics simulations and time-dependent fluorescent shift analyses, we unravelled individual sensitivities of these probes to different biophysical properties, their distinct localizations and specific relaxation processes in membranes. Overall, Pro12A, NR12S and NR12A serve together as a toolbox with a wide range of applications allowing to select the most appropriate probe for each specific research question.

Rapid detection of hydrogen sulfide in vegetables and monosodium glutamate based on perylene supramolecular aggregates using an indicator displacement assays strategy.

Hydrogen sulfide (H2S) has been clearly identified as a hazardous chemical pollutant that seriously affects food safety and human health. In order to develop a rapid, accurate and efficient H2S tracking method, this work propose a strategy based on indicator displacement assays (IDA). A water-soluble histidine-modified perylene diimide fluorescent probe was synthesized by a one-step method, and the probe can form supramolecular aggregates in the presence of Cd2+. There will be a fluorescence transformation of probe, caused by the change of the state of aggregation and adjusted by various concentration of S2-, which can achieve the fluorescence detection of S2-. The limit of detection is as low as 0.41 µmol/L. Particularly worth mentioning is that the probe in this work can be recycled for at least 5 times, which is environmentally friendly and economical. Finally, this method was applied in three kinds of vegetables and monosodium glutamate samples.

Assessment of Photoreleasable Linkers and Light-Capturing Antennas on a Photoresponsive Cobalamin Scaffold.

Cobalamin has shown promise as a light-sensitive drug delivery platform owing to its ease of modification and the high quantum yields for drug photorelease. However, studies to date on the general photochemistry of alkyl cobalamins have primarily focused on methyl and adenosyl-substituted derivatives, the natural cofactors present in various enzymatic species. We describe the synthesis and photolytic behavior of cobalamin conjugates comprised of different combinations of fluorophores and ß-axial ligands. In general, cobalamin conjugates containing ß-axial alkyl substituents undergo efficient photolysis under aqueous conditions, with quantum yields up to >40%. However, substituents that are large and hydrophobic, or unable to readily support the presumed radical intermediate, suffer less efficient photolysis (<15%) than smaller, water-soluble, analogs. By contrast, quantum yields improve by 2-fold in DMF for cobalamins containing large hydrophobic ß-axial substituents. This suggests that drug release from carriers comprised of membranous compartments, such as liposomes, may be significantly more efficient than the corresponding photorelease in an aqueous environment. Finally, we explored the impact of fluorophores on the photolysis of alkyl cobalamins under tissue-mimetic conditions. Cobalamins substituted with efficient photon-capturing fluorophores display up to 4-fold enhancements in photolysis relative to unsubstituted derivatives. In summary, we have shown that the photosensitivity of alkyl cobalamin conjugates can be tuned by altering the Co-appended alkyl moiety, modulating the polarity of the environment (solvent), and installing photon-capturing fluorophores onto the cobalamin framework.

Assessment of Intracellular GTP Levels Using Genetically Encoded Fluorescent Sensors.

Changes in intracellular GTP levels, even incremental ones, profoundly affect the activity of several GTP-binding proteins ultimately resulting in alteration of several basal cellular phenotypes including cell motility, invasion, and tumorigenesis. However, until recently, no tools were available for GTP quantification in live cells. Therefore, in the current chapter, we describe the methodology for the quantitative assessment of spatiotemporal changes in GTP levels in the cells using genetically encoded fluorescent ratiometric GTP sensors termed GEVALs for GTP evaluators.

Fricke gel xylenol orange dosimeter layers for stereotactic radiosurgery: A preliminary approach.

Investigations regarding the feasibility, reliability, and accuracy of Fricke gel dosimeter layers for stereotactic radiosurgery are presented. A representative radiosurgery plan consisting of two targets has been investigated. Absorbed dose distributions measured using radiochromic films and gelatin Fricke Gel dosimetry in layers have been compared with dose distributions calculated by using a treatment planning system and Monte Carlo simulations. The different dose distributions have been compared by means of the gamma index demonstrating that gelatin Fricke gel dosimeter layers showed agreements of 100%, 100%, and 93%, with dose and distance tolerances of 2% and 2 mm, with respect to film dosimetry, treatment planning system and Monte Carlo simulations, respectively. The capability of the developed system for three-dimensional dose mapping was shown, obtaining promising results when compared with well-established dosimetry methods. The obtained results support the viability of Fricke gel dosimeter layers analyzed by optical methods for stereotactic radiosurgery.

The Mean Single Molecule Rate (mSMR) in the Analysis of Fluorescence Fluctuations: Measurements on DNA Mixtures of Defined Composition.

We present a method for the evaluation of fluorescence fluctuations on the basis of Mandel's Q parameter, using sampling time-dependent factorial cumulants. By relating the Q parameter to the sampling time, we obtain the mean single molecule rate (mSMR), an easy to interpret expression that provides both brightness and diffusion information. The model is suitable for the widely used confocal setups with single photon excitation and a single detection channel. We present a way to correct the mSMR for afterpulsing, dead time and background noise. To account for photokinetic effects at short sampling times, we expand the model by a simple on/off isomerization term, which is similar to the well-known triplet model. The functionality of the mSMR is shown using Monte Carlo simulations. The correction mechanisms and the experimental applicability of the model are then demonstrated by DNA measurements of defined composition. By systematically analyzing DNA mixtures, we can show that at large sampling times, the mSMR correctly describes the single molecule brightness rates and the diffusive properties of DNA molecules. At short sampling times, the photokinetic effects of isomerization are accurately described by the mSMR model. Since additionally the mSMR can easily be corrected for measurement artefacts such as detector dead time, afterpulsing and background noise, this is a valuable advantage over the standard method of fluorescence correlation spectroscopy.