A novel pH-activated AIEgen probe for dynamic lysosome tracking and high-efficiency photodynamic therapy.

A novel AIEgen molecular probe (N-3QL) with typical AIE effects, good biocompatibility, lysosome targeting, pH activation, excellent photostability, and high brightness was synthesized using two simple synthetic steps. Spectroscopic and cytotoxicity experiments indicate that N-3QL can not only be used for the dynamic monitoring of cancer cell lysosomes, but also for photodynamic therapy (PDT) ablation of cancer cells.

Molecular to Supramolecular Self-Assembled Luminogens for Tracking the Intracellular Organelle Dynamics.

Deciphering the dynamics of intracellular organelles has gained immense attention due to their subtle control over diverse, complex biological processes such as cellular metabolism, energy homeostasis, and autophagy. In this context, molecular materials, including small-organic fluorescent probes and their supramolecular self-assembled nano-/microarchitectures, have been employed to explore the diverse intracellular biological events. However, only a handful of fluorescent probes and self-assembled emissive structures have been successfully used to track different organelle's movements, circumventing the issues related to water solubility and long-term photostability. Thus, the water-soluble molecular fluorescent probes and the water-dispersible supramolecular self-assemblies have emerged as promising candidates to explore the trafficking of the organelles under diverse physiological conditions. In this review, we have delineated the recent progress of fluorescent probes and their supramolecular self-assemblies for the elucidation of the dynamics of diverse cellular organelles with a special emphasis on lysosomes, lipid droplets, and mitochondria. Recent advancement in fluorescence lifetime and super-resolution microscopy imaging has also been discussed to investigate the dynamics of organelles. In addition, the fabrication of the next-generation molecular to supramolecular self-assembled luminogens for probing the variation of microenvironments during the trafficking process has been outlined.

Lab-on-a-Molecule Probe: Multitarget Detection of Five Aromatic Pesticides Using a Supramolecular Probe under Single Wavelength Excitation.

In order to prevent and control the effects of pesticide residues on human health and the ecological environment, the rapid, highly sensitive, and selective detection of multiple pesticide residues has become an urgent problem to be solved. Herein, a lab-on-a-molecule probe based on a host-guest complex (ThT@Q[8] probe) has been developed to simultaneously analyze multiple aromatic pesticides under single wavelength excitation, such as fuberidazole, thiabendazole, carbendazim, thidiazuron, and tricyclazole. The fluorescence titration spectra of the ThT@Q[8] probe with the five pesticides mentioned above showed that the fluorescence intensity exhibited a good linear correlation with the pesticide concentration and the limit of detection was as low as 10-7 M. Because the ThT@Q[8] probe exhibits diverse fluorescence color changes to the five pesticides studied under a 365 nm ultraviolet lamp, we fabricated a single probe used to detect multiple analytes in the RGB triple channel by extracting the RGB variations. Principal component analysis and linear discriminant analysis proved that the ThT@Q[8] probe can recognize and distinguish five pesticides and can be applied at different concentrations. In real samples, the ThT@Q[8] probe recognized and distinguished five pesticides in tap water and Huaxi River water. The 1H NMR spectra results proved that a charge-transfer complex of ThT and pesticides in the Q[8] cavity may be formed. Moreover, we selected a test strip as a carrier to detect pesticides. The results indicate it can be used to quickly and conveniently detect different pesticides due to the rapid color change. Besides, the ThT@Q[8] probe has good cell permeability and can be used to detect pesticide residues in living cells. This work has laid the foundation for the qualitative and quantitative multitarget detection of pesticide residues.

Recent advances of fluorescent biosensors based on cyclic signal amplification technology in biomedical detection.

The cyclic signal amplification technology has been widely applied for the ultrasensitive detection of many important biomolecules, such as nucleic acids, proteins, enzymes, adenosine triphosphate (ATP), metal ions, exosome, etc. Due to their low content in the complex biological samples, traditional detection methods are insufficient to satisfy the requirements for monitoring those biomolecules. Therefore, effective and sensitive biosensors based on cyclic signal amplification technology are of great significance for the quick and simple diagnosis and treatment of diseases. Fluorescent biosensor based on cyclic signal amplification technology has become a research hotspot due to its simple operation, low cost, short time, high sensitivity and high specificity. This paper introduces several cyclic amplification methods, such as rolling circle amplification (RCA), strand displacement reactions (SDR) and enzyme-assisted amplification (EAA), and summarizes the research progress of using this technology in the detection of different biomolecules in recent years, in order to provide help for the research of more efficient and sensitive detection methods.

Renal-Clearable Molecular Probe for Near-Infrared Fluorescence Imaging and Urinalysis of SARS-CoV-2.

Despite the importance of rapid and accurate detection of SARS-CoV-2 in controlling the COVID-19 pandemic, current diagnostic methods are static and unable to distinguish between viable/nonviable virus or directly reflect viral replication activity. Real-time imaging of protease activity specific to SARS-CoV-2 can overcome these issues but remains lacking. Herein, we report a near-infrared fluorescence (NIRF) activatable molecular probe (SARS-CyCD) for detection of SARS-CoV-2 protease in living mice. The probe comprises a hemicyanine fluorophore caged with a protease peptide substrate and a cyclodextrin unit, which function as an NIRF signaling moiety and a renal-clearable enabler, respectively. The peptide substrate of SARS-CyCD can be specifically cleaved by SARS-CoV-2 main protease (Mpro), resulting in NIRF signal activation and liberation of the renal-clearable fluorescent fragment (CyCD). Such a design not only allows sensitive detection of Mpro in the lungs of living mice after intratracheal administration but also permits optical urinalysis of SARS-CoV-2 infection. Thus, this study presents an in vivo sensor that holds potential in preclinical high-throughput drug screening and clinical diagnostics for respiratory viral infections.

Profiling sirtuin activity using Copper-free click chemistry.

The mammalian sirtuins are a group of posttranslational modification enzymes that remove acyl modifications from lysine residues in an NAD+-dependent manner. Although initially proposed as histone deacetylases (HDACs), they are now known to target other cellular enzymes and proteins as well. Sirtuin-catalyzed simple amide hydrolysis has profound biological consequences including suppression of gene expression, promotion of DNA damage repair, and regulation of glucose and lipid metabolism. Human sirtuins have been intensively pursued by both academia and industry as potential therapeutic targets for the treatment of diseases such as cancer and neurodegeneration. To gain a better understanding of their roles in various cellular events, innovative chemical probes are highly sought after. This current study focuses on the development of activity-based chemical probes (ABPs) for the profiling of sirtuin activity in biological samples. Cyclooctyne-containing and azido-containing probes were synthesized to enable the subsequent copper-free "click" conjugation to either a fluorophore or biotin. The two groups of structurally related ABPs demonstrated different labeling efficiency and selectivity: the cyclooctyne-containing probes failed to label recombinant sirtuins to any appreciable level, while the azido-containing ABPs showed good isoform selectivity. The azido-containing ABPs were further analyzed for their ability to label an individual sirtuin isoform in protein mixtures and cell lysates. These biocompatible ABPs allow the study of dynamic cellular protein activity change to become possible.

Mechanical stimulus-evoked signal transduction between keratinocytes and sensory neurons via extracellular ATP.

The skin is exposed to various external stimuli. Keratinocytes, which are the main cell type in the epidermis, interact with peripheral sensory neurons and modulate neuronal activity. Recent studies have revealed that keratinocytes play crucial roles in nociception, and that ATP is one of the main mediators of signal transduction from keratinocytes to sensory neurons. However, no quantitative cellular level analyses of ATP-mediated information flow from keratinocytes to sensory dorsal root ganglion (DRG) neurons have been conducted. In this study, we performed simultaneous imaging of cell surface ATP and intracellular Ca2+ signals using both iATPSnFR, a genetically encoded ATP probe localized to the outside of the cell membrane, and the Ca2+ probe, Fura-red. Upon mechanical stimulation of the keratinocyte with a glass needle, an increase in Ca2+ and ATP release were observed around the stimulated area, and these phenomena were positively correlated. In cultured DRG neurons and keratinocytes neighboring the stimulated keratinocyte, increased intracellular Ca2+ concentration and levels of cell surface ATP on the side closer to the stimulated cell were detected. The ratio of Ca2+ response to input ATP signal was significantly larger in DRG neurons than in keratinocytes. We found that DRG neurons were more sensitive to ATP than keratinocytes, and therefore, only DRG neurons responded to ATP at 1 µM or lower concentrations when in co-culture with keratinocytes. Moreover, signals caused by moderate mechanical stimulation of keratinocytes were transmitted predominantly to DRG neurons. These findings would be important in the further determination of the detailed mechanism of nociception in the epidermis.

3D positioning of tagged DNA loci by widefield and super-resolution fluorescence imaging of fixed yeast nuclei.

This protocol describes how to culture, image, and determine the nuclear position of a fluorescently tagged DNA locus in the 3D nucleoplasm of fixed Saccharomyces cerevisiae cells. Here, we propose a manual scoring method based on widefield images and an automated method based on 3D-SIM images. Yeast culture conditions have to be followed meticulously to get the best biological response in a given environment. For complete details on the use and execution of this protocol, please refer to Forey et al. (2020).

Protocol for synthesis and use of a turn-on fluorescent probe for quantifying labile Zn2+ in the Golgi apparatus in live cells.

Quantitative analysis using a turn-on fluorescent probe is inherently difficult due to the dependency of the fluorescence intensity on the probe concentration. To overcome this limitation, we developed an in situ quantification method using a turn-on fluorescent probe and a standard fluorophore, which are colocalized by protein tag technology. This protocol describes the synthesis of a Zn2+ probe, named ZnDA-1H, and the procedure to quantify the labile Zn2+ concentration in the Golgi of live HeLa cells by confocal fluorescence microscopy. For complete details on the use and execution of this protocol, please refer to Kowada et al. (2020).

Quantitative imaging of intracellular nanoparticle exposure enables prediction of nanotherapeutic efficacy.

Nanoparticle internalisation is crucial for the precise delivery of drug/genes to its intracellular targets. Conventional quantification strategies can provide the overall profiling of nanoparticle biodistribution, but fail to unambiguously differentiate the intracellularly bioavailable particles from those in tumour intravascular and extracellular microenvironment. Herein, we develop a binary ratiometric nanoreporter (BiRN) that can specifically convert subtle pH variations involved in the endocytic events into digitised signal output, enabling the accurately quantifying of cellular internalisation without introducing extracellular contributions. Using BiRN technology, we find only 10.7-28.2% of accumulated nanoparticles are internalised into intracellular compartments with high heterogeneity within and between different tumour types. We demonstrate the therapeutic responses of nanomedicines are successfully predicted based on intracellular nanoparticle exposure rather than the overall accumulation in tumour mass. This nonlinear optical nanotechnology offers a valuable imaging tool to evaluate the tumour targeting of new nanomedicines and stratify patients for personalised cancer therapy.

1,3,5-Triaza-7-Phosphaadamantane (PTA) as a 31P NMR Probe for Organometallic Transition Metal Complexes in Solution.

Due to the rigid structure of 1,3,5-triaza-7-phosphaadamantane (PTA), its 31P chemical shift solely depends on non-covalent interactions in which the molecule is involved. The maximum range of change caused by the most common of these, hydrogen bonding, is only 6 ppm, because the active site is one of the PTA nitrogen atoms. In contrast, when the PTA phosphorus atom is coordinated to a metal, the range of change exceeds 100 ppm. This feature can be used to support or reject specific structural models of organometallic transition metal complexes in solution by comparing the experimental and Density Functional Theory (DFT) calculated values of this 31P chemical shift. This approach has been tested on a variety of the metals of groups 8-12 and molecular structures. General recommendations for appropriate basis sets are reported.

Detection of bacterial colonization by the spectral changes of surface-enhanced Raman reporters.

In times of widespread multiple antibiotic resistance, the bacterial colonization of crucial medical surfaces should be detected as fast as possible. In this work, we present the non-destructive SERS method for the detection of bacterial colonization. SERS is an excellent tool for the monitoring of suitable substances in low concentrations. The SERS substrate was prepared by the aggregation of citrate-stabilized gold nanoparticles and the adsorption of the reporters (crystal violet, thiamine, and adenine). We have tested the substrate for the detection of clinically relevant S. aureus and P. aeruginosa bacteria. The SERS spectra before and after the substrate incubation revealed the degradation of the reporter by the growing bacteria. The growth of P. aeruginosa was detected using the substrates with preadsorbed crystal violet or adenine. The suitable reporter for the detection of S. aureus remains to be discovered. The selection of the reporters resistant to exposure but easily degraded by bacteria will open the way for the in situ monitoring of bacterial colonization, thus complementing the arsenal of methods in the battle against hospital infections.

Assessing The Key Photophysical Properties of Triangulenium Dyes for DNA Binding by Alteration of the Fluorescent Core.

Four-stranded G-quadruplex (G4) DNA is a non-canonical DNA topology that has been proposed to form in cells and play key roles in how the genome is read and used by the cellular machinery. Previously, a fluorescent triangulenium probe (DAOTA-M2) was used to visualise G4s in cellulo, thanks to its distinct fluorescence lifetimes when bound to different DNA topologies. Herein, the library of available triangulenium probes is expanded to explore how modifications to the fluorescent core of the molecule affect its photophysical characteristics, interaction with DNA and cellular localisation. The benzo-bridged and isopropyl-bridged diazatriangulenium dyes, BDATA-M2 and CDATA-M2 respectively, featuring ethyl-morpholino substituents, were synthesised and characterised. The interactions of these molecules with different DNA topologies were studied to determine their binding affinity, fluorescence enhancement and fluorescence lifetime response. Finally, the cellular uptake and localisation of these optical probes were investigated. Whilst structural modifications to the triangulenium core only slightly alter the binding affinity to DNA, BDATA-M2 and CDATA-M2 cannot distinguish between DNA topologies through their fluorescence lifetime. It is argued theoretically and experimentally that this is due to reduced effectiveness of photoinduced electron transfer (PET) quenching. This work presents valuable new evidence into the critical role of PET quenching when using the fluorescence lifetime of triangulenium dyes to discriminate G4 DNA from duplex DNA, highlighting the importance of fine tuning redox and spectral properties when developing new triangulenium-based G4 probes.

The chromatographic behaviour of new double-labelled oligodeoxynucleotide probes containing azaphthalocyanine dye as a quencher with respect to evaluation of their purity.

The influence of experimental conditions on chromatographic behaviour of promising oligodeoxynucleotide double-labelled molecular probes containing an azaphthalocyanine macrocycle as a perspective dark quencher was studied. A recently introduced new stationary phase based on styrene-divinylbenzene copolymer was tested. The planar and hydrophobic structure of the azaphthalocyanine is considerably different from those of currently used fluorophores and quenchers. Thus, the most challenging issue was the separation of the double-labelled probe from its main impurity represented by a mono-labelled probe, containing only the azaphthalocyanine macrocycle. The absorbance measurement cannot simply determine this impurity, and its presence fundamentally compromises the biological assay. The commonly used gradient elution was not suitable and isocratic conditions seemed to be more appropriate. The azaphthalocyanine moiety influences the properties of the modified oligodeoxynucleotides substantially, and thus their chromatographic behaviour was determined predominantly by this quencher. Acetonitrile was the preferred organic solvent for the analysis of probes containing the azaphthalocyanine quencher and the effect of ion-pairing reagents was dependent on the probe structure. The temperature seemed to be an effective parameter for fine-tuning of the separation and mass transfer improvement. Generally, our findings could be helpful in method development for purity evaluation of double-labelled oligodeoxynucleotide probes and semipreparative methods.

DNA-Based Nanostructures for Live-Cell Analysis.

DNA-based probes constitute a versatile platform for making biological measurements due to their ability to recognize both nucleic acid and non-nucleic acid targets, ease of synthesis and chemical modification, amenability to be interfaced with signal amplification schemes, and inherent biocompatibility. Here, we provide a historical perspective of how a transition from linear DNA structures toward more structurally complex nanostructures has revolutionized live-cell analysis. Modulating the structure gives rise to probes that can enter cells without the aid of transfection reagents and can detect, track, and quantify analytes in live cells at the single-organelle, single-cell, tissue section, and whole organism levels. We delineate the advantages and disadvantages associated with different probe architectures and describe the advances enabled by these structures for elucidating fundamental biology as well as developing improved diagnostic and theranostic systems. We also discuss the outstanding challenges in the field and outline potential solutions.

Imaging of peroxynitrite in drug-induced acute kidney injury with a near-infrared fluorescence and photoacoustic dual-modal molecular probe.

A FRET-based probe for mapping the fluctuation of ONOO- in cisplatin-induced acute kidney injury was constructed. It exhibits ratiometric near infrared fluorescence and a dramatic decrease of its peak absorbance at 719 nm upon addition of ONOO- that is converted into remarkable signal changes in fluorescence and photoacoustic images respectively.

A general strategy exploiting m5C duplex-remodelling effect for selective detection of RNA and DNA m5C methyltransferase activity in cells.

RNA:5-methylcytosine (m5C) methyltransferases are currently the focus of intense research following a series of high-profile reports documenting their physiological links to several diseases. However, no methods exist which permit the specific analysis of RNA:m5C methyltransferases in cells. Herein, we described how a combination of biophysical studies led us to identify distinct duplex-remodelling effects of m5C on RNA and DNA duplexes. Specifically, m5C induces a C3'-endo to C2'-endo sugar-pucker switch in CpG RNA duplex but triggers a B-to-Z transformation in CpG DNA duplex. Inspired by these different 'structural signatures', we developed a m5C-sensitive probe which fluoresces spontaneously in response to m5C-induced sugar-pucker switch, hence useful for sensing RNA:m5C methyltransferase activity. Through the use of this probe, we achieved real-time imaging and flow cytometry analysis of NOP2/Sun RNA methyltransferase 2 (NSUN2) activity in HeLa cells. We further applied the probe to the cell-based screening of NSUN2 inhibitors. The developed strategy could also be adapted for the detection of DNA:m5C methyltransferases. This was demonstrated by the development of DNA m5C-probe which permits the screening of DNA methyltransferase 3A inhibitors. To our knowledge, this study represents not only the first examples of m5C-responsive probes, but also a new strategy for discriminating RNA and DNA m5C methyltransferase activity in cells.