Signal-enhanced real-time magnetic resonance of enzymatic reactions at millitesla fields

The phenomenon of nuclear magnetic resonance (NMR) is widely applied in biomedical and biological science to study structures and dynamics of proteins and their reactions. Despite its impact, NMR is an inherently insensitive phenomenon and has driven the field to construct spectrometers with increasingly higher magnetic fields leading to more detection sensitivity. Here, we are demonstrating that enzymatic reactions can be followed in real-time at millitesla fields, three orders of magnitude lower than the field of state-of-the-art NMR spectrometers. This requires signal-enhancing samples via hyperpolarization. Within seconds, we have enhanced the signals of 2-¹³C-pyruvate, an important metabolite to probe cancer metabolism, in 22 mM concentrations (up to 10.1% ± 0.1% polarization) and show that such a large signal allows for the real-time detection of enzymatic conversion of pyruvate to lactate at 24 mT. This development paves the pathways for biological studies in portable and affordable NMR systems with a potential for medical diagnostics.

We demonstrate that metabolism can be monitored in real-time with magnetic resonance at milli-tesla fields that are 1000 fold lower than state-of-the-art high field spectrometers.     

Quantifying tensile forces at cell–cell junctions with a DNA-based fluorescent probe

Cells are physically contacting with each other. Direct and precise quantification of forces at cell–cell junctions is still challenging. Herein, we have developed a DNA-based ratiometric fluorescent probe, termed DNAMeter, to quantify intercellular tensile forces. These lipid-modified DNAMeters can spontaneously anchor onto live cell membranes. The DNAMeter consists of two self-assembled DNA hairpins of different force tolerance. Once the intercellular tension exceeds the force tolerance to unfold a DNA hairpin, a specific fluorescence signal will be activated, which enables the real-time imaging and quantification of tensile forces. Using E-cadherin-modified DNAMeter as an example, we have demonstrated an approach to quantify, at the molecular level, the magnitude and distribution of E-cadherin tension among epithelial cells. Compatible with readily accessible fluorescence microscopes, these easy-to-use DNA tension probes can be broadly used to quantify mechanotransduction in collective cell behaviors.

A DNA-based fluorescent probe to quantify the magnitude and distribution of tensile forces at cell–cell junctions.     

Time-resolved luminescence detection of peroxynitrite using a reactivity-based lanthanide probe

Peroxynitrite (ONOO⁻) is a powerful and short-lived oxidant formed in vivo, which can react with most biomolecules directly. To fully understand the roles of ONOO⁻ in cell biology, improved methods for the selective detection and real-time analysis of ONOO⁻ are needed. We present a water-soluble, luminescent europium(iii) probe for the rapid and sensitive detection of peroxynitrite in human serum, living cells and biological matrices. We have utilised the long luminescence lifetime of the probe to measure ONOO⁻ in a time-resolved manner, effectively avoiding the influence of autofluorescence in biological samples. To demonstrate the utility of the Eu(iii) probe, we monitored the production of ONOO⁻ in different cell lines, following treatment with a cold atmospheric plasma device commonly used in the clinic for skin wound treatment.

Reactivity-based europium(iii) probe displays excellent selectivity for peroxynitrite (ONOO⁻), enabling its time-resolved luminescence detection in living cells.     

A new pentacyclic pyrylium fluorescent probe that responds to pH imbalance during apoptosis

Efficient fluorophores with easy synthetic routes and fast responses are of great importance in clinical diagnostics. Herein, we report a new, rigid pentacyclic pyrylium fluorophore, PS-OMe, synthesised in a single step by a modified Vilsmeier–Haack reaction. Insights into the reaction mechanism facilitated a new reaction protocol for the efficient synthesis of PS-OMe which upon demethylation resulted in a “turn-on” pH sensor, PS-OH. This new fluorescent probe has been successfully used to monitor intracellular acidification at physiological pH. From the fluorescence image analysis, we were able to quantify the intracellular dynamic pH change during apoptosis. This new pH probe is a potential chemical tool for screening, drug discovery and dose determination in cancer therapy.

A modified Vilsmeier–Haack reaction resulted in the synthesis of a pyrylium based turn-on fluorescent pH probe. The probe can monitor minute acidification and dynamic pH variation in cells during apoptosis with therapeutic chemo drugs.     

A pyrone remodeling strategy to access diverse heterocycles: application to the synthesis of fascaplysin natural products

The synthesis of diverse N-fused heterocycles, including the pyrido[1,2-a]indole scaffold, using an efficient pyrone remodeling strategy is described. The pyrido[1,2-a]indole core was demonstrated to be a versatile scaffold that can be site-selectively functionalized. The utility of this novel annulation strategy was showcased in a concise formal synthesis of three fascaplysin congeners.

The synthesis of diverse N-fused heterocycles, including the pyrido[1,2-a]indole scaffold, using an efficient pyrone remodeling strategy is described.     

Self-assembled amphiphilic fluorescent probe: detecting pH-fluctuations within cancer cells and tumour tissues

Abnormal anaerobic metabolism leads to a lowering of the pH of many tumours, both within specific intracellular organelles and in the surrounding extracellular regions. Information relating to pH-fluctuations in cells and tissues could aid in the identification of neoplastic lesions and in understanding the determinants of carcinogenesis. Here we report an amphiphilic fluorescent pH probe (CS-1) that, as a result of its temporal motion, provides pH-related information in cancer cell membranes and selected intracellular organelles without the need for specific tumour targeting. Time-dependent cell imaging studies reveal that CS-1 localizes within the cancer cell-membrane about 20 min post-incubation. This is followed by migration to the lysosomes at 30 min before being taken up in the mitochondria after about 60 min. Probe CS-1 can selectively label cancer cells and 3D cancer spheroids and be readily observed using the green fluorescence channel (λ_em = 532 nm). In contrast, CS-1 only labels normal cells marginally, with relatively low Pearson''s correlation coefficients being found when co-incubated with standard intracellular organelle probes. Both in vivo and ex vivo experiments provide support for the suggestion that CS-1 acts as a fluorescent label for the periphery of tumours, an effect ascribed to proton-induced aggregation. A much lower response is seen for muscle and liver. Based on the present results, we propose that sensors such as CS-1 may have a role to play in the clinical and pathological detection of tumour tissues or serve as guiding aids for surgery.

A self-assembled amphiphilic fluorescent probe allows pH-fluctuations within cancer cells and tumour tissues to be readily detected.     

One stone,three birds: one AIEgen with three colors for fast differentiation of three pathogens

Visually identifying pathogens favors rapid diagnosis at the point-of-care testing level. Here, we developed a microenvironment-sensitive aggregation-induced emission luminogen (AIEgen), namely IQ–Cm, for achieving fast discrimination of Gram-negative bacteria, Gram-positive bacteria and fungi by the naked-eye. With a twisted donor–acceptor and multi-rotor structure, IQ–Cm shows twisted intramolecular charge transfer (TICT) and AIE properties with sensitive fluorescence color response to the microenvironment of pathogens. Driven by the intrinsic structural differences of pathogens, IQ–Cm with a cationic isoquinolinium moiety and a membrane-active coumarin unit as the targeting and interacting groups selectively locates in different sites of three pathogens and gives three naked-eye discernible emission colors. Gram-negative bacteria are weak pink, Gram-positive bacteria are orange-red and fungi are bright yellow. Therefore, based on their distinctive fluorescence response, IQ–Cm can directly discriminate the three pathogens at the cell level under a fluorescence microscope. Furthermore, we demonstrated the feasibility of IQ–Cm as a visual probe for fast diagnosis of urinary tract infections, timely monitoring of hospital-acquired infection processes and fast detection of molds in the food field. This simple visualization strategy based on one single AIEgen provides a promising platform for rapid pathogen detection and point-of-care diagnosis.

A simple AIEgen with three emission colors achieves rapid identification of Gram-negative bacteria, Gram-positive bacteria and fungi.     

Protein encapsulation: a new approach for improving the capability of small-molecule fluorogenic probes

Herein, we report a protein-based hybridization strategy that exploits the host-guest chemistry of HSA (human serum albumin) to solubilize the otherwise cell impermeable ONOO⁻ fluorescent probe Pinkment-OAc. Formation of a HSA/Pinkment-OAc supramolecular hybrid was confirmed by SAXS and solution-state analyses. This HSA/Pinkment-OAc hybrid provided an enhanced fluorescence response towards ONOO⁻versusPinkment-OAc alone, as determined by in vitro experiments. The HSA/Pinkment-OAc hybrid was also evaluated in RAW 264.7 macrophages and HeLa cancer cell lines, which displayed an enhanced cell permeability enabling the detection of SIN-1 and LPS generated ONOO⁻ and the in vivo imaging of acute inflammation in LPS-treated mice. A remarkable 5.6 fold (RAW 264.7), 8.7-fold (HeLa) and 2.7-fold increased response was seen relative to Pinkment-OAc alone at the cellular level and in vivo, respectively. We anticipate that HSA/fluorescent probe hybrids will soon become ubiquitous and routinely applied to overcome solubility issues associated with hydrophobic fluorescent imaging agents designed to detect disease related biomarkers.

Herein, we report a protein-based hybridization strategy that exploits the host–guest chemistry of HSA (human serum albumin) to solubilize the otherwise cell impermeable ONOO⁻ fluorescent probe Pinkment-OAc.     

Probing biotin receptors in cancer cells with rationally designed fluorogenic squaraine dimers

Fluorogenic probes enable imaging biomolecular targets with high sensitivity and maximal signal-to-background ratio under non-wash conditions. Here, we focus on the molecular design of biotinylated dimeric squaraines that undergo aggregation-caused quenching in aqueous media through intramolecular H-type dimerization, but turn on their fluorescence in apolar environment due to target-mediated disaggregation. Our structure–property study revealed that depending on the linkers used to connect the squaraine dyes, different aggregation patterns could be obtained (intramolecular dimerization versus intermolecular aggregation) leading to different probing efficiencies. Using a relatively short l-lysine linker we developed a bright fluorogenic probe, Sq2B, displaying only intramolecular dimerization-caused quenching properties in aqueous media. The latter was successfully applied for imaging biotin receptors, in particular sodium-dependent multivitamin transporter (SMVT), which are overexpressed at the surface of cancer cells. Competitive displacement with SMVT-targets, such as biotin, lipoic acid or sodium pantothenate, showed Sq2B targeting ability to SMVT. This fluorogenic probe for biotin receptors could distinguish cancer cells (HeLa and KB) from model non-cancer cell lines (NIH/3T3 and HEK293T). The obtained results provide guidelines for development of new dimerization-based fluorogenic probes and propose bright tools for imaging biotin receptors, which is particularly important for specific detection of cancer cells.

Rational design of self-quenched squaraine dimers bearing biotin yielded a bright fluorogenic probe that can distinguish cancerous from non-cancerous cells.     

Multiplexed droplet loop-mediated isothermal amplification with scorpion-shaped probes and fluorescence microscopic counting for digital quantification of virus RNAs

Highly sensitive digital nucleic acid techniques are of great significance for the prevention and control of epidemic diseases. Here we report the development of multiplexed droplet loop-mediated isothermal amplification (multiplexed dLAMP) with scorpion-shaped probes (SPs) and fluorescence microscopic counting for simultaneous quantification of multiple targets. A set of target-specific fluorescence-activable SPs are designed, which allows establishment of a novel multiplexed LAMP strategy for simultaneous detection of multiple cDNA targets. The digital multiplexed LAMP assay is thus developed by implementing the LAMP reaction using a droplet microfluidic chip coupled to a droplet counting microwell chip. The droplet counting system allows rapid and accurate counting of the numbers of total droplets and the positive droplets by collecting multi-color fluorescence images of the droplets in a microwell. The multiplexed dLAMP assay was successfully demonstrated for the quantification of HCV and HIV cDNA with high precision and detection limits as low as 4 copies per reaction. We also verified its potential for simultaneous digital assay of HCV and HIV RNA in clinical plasma samples. This multiplexed dLAMP technique can afford a useful platform for highly sensitive and specific detection of nucleic acids of viruses and other pathogens, enabling rapid diagnosis and prevention of infectious diseases.

The development of multiplexed dLAMP with scorpion-shaped probes and fluorescence microscopic counting affords simultaneous digital quantification of multiple virus RNAs.     

Persistent organic room temperature phosphorescence: what is the role of molecular dimers?

Molecular dimers have been frequently found to play an important role in room temperature phosphorescence (RTP), but its inherent working mechanism has remained unclear. Herein a series of unique characteristics, including singlet excimer emission and thermally activated delayed fluorescence, were successfully integrated into a new RTP luminogen of CS-2COOCH₃ to clearly reveal the excited-state process of RTP and the special role of molecular dimers in persistent RTP emission.

The first purely organic room temperature phosphorescence (RTP) luminogen, with singlet excimer emission and thermally activated delayed fluorescence (TADF) effect, was successfully developed.      

Rapid and ultrasensitive electrochemical detection of circulating tumor DNA by hybridization on the network of gold-coated magnetic nanoparticles

An accurate and robust method for quantifying the levels of circulating tumor DNA (ctDNA) is vital if this potential biomarker is to be used for the early diagnosis of cancer. The analysis of ctDNA presents unique challenges because of its short half-life and ultralow abundance in early stage cancers. Here we develop an ultrasensitive electrochemical biosensor for rapid detection of ctDNA in whole blood. The sensing of ctDNA is based on hybridization on a network of probe DNA modified gold-coated magnetic nanoparticles (DNA-Au@MNPs). This DNA-Au@MNPs biosensor can selectively detect short- and long-strand DNA targets. It has a broad dynamic range (2 aM to 20 nM) for 22 nucleotide DNA target with an ultralow detection limit of 3.3 aM. For 101 nucleotide ctDNA target, a dynamic range from 200 aM to 20 nM was achieved with a detection limit of 5 fM. This DNA-Au@MNPs based sensor provides a promising method to achieve 20 min response time and minimally invasive cancer early diagnosis.

This study introduces a new electrochemical sensing strategy for the rapid detection of circulating tumor DNA (ctDNA) from whole blood in combination with a network of DNA-Au@MNPs with high sensitivity and excellent selectivity.     

In vivo monitoring of tissue regeneration using a ratiometric lysosomal AIE probe

Tissue regeneration is a crucial self-renewal capability involving many complex biological processes. Although transgenic techniques and fluorescence immunohistochemical staining have promoted our understanding of tissue regeneration, simultaneous quantification and visualization of tissue regeneration processes is not easy to achieve. Herein, we developed a simple and quantitative method for the real-time and non-invasive observation of the process of tissue regeneration. The synthesized ratiometric aggregation-induced-emission (AIE) probe exhibits high selectivity and reversibility for pH responses, good ability to map lysosomal pH both in vitro and in vivo, good biocompatibility and excellent photostability. The caudal fin regeneration of a fish model (medaka larvae) was monitored by tracking the lysosomal pH change. It was found that the mean lysosomal pH is reduced during 24–48 hpa to promote the autophagic activity for cell debris degradation. Our research can quantify the changes in mean lysosomal pH and also exhibit its distribution during the caudal fin regeneration. We believe that the AIE-active lysosomal pH probe can also be potentially used for long-term tracking of various lysosome-involved biological processes, such as tracking the stress responses of tissue, tracking the inflammatory responses, and so on.

An AIE-active ratiometric probe for the first time achieved the long-term quantification of lysosomal pH during the medaka larva''s caudal fin regeneration.     

Fluorogenic probes for detecting deacylase and demethylase activity towards post-translationally-modified lysine residues

Reversible enzymatic post-translational modification of the ε-amino groups of lysine residues (e.g. N-acylation reactions) plays an important role in regulating the cellular activities of numerous proteins. This study describes how enzyme catalyzed N-deprotection of lysine residues of non-fluorescent peptide-coumarin probes can be used to generate N-deprotected peptides that undergo spontaneous O- to N-ester transfer reactions (uncatalyzed) to generate a highly fluorescent N-carbamoyl peptide. This enables detection of enzyme catalyzed N-deacetylation, N-demalonylation, N-desuccinylation and N-demethylation reactions activities towards the N-modified lysine residues of these probes using simple ‘turn on’ fluorescent assays.

We developed “turn-on” fluorescent probes that detect enzymatic lysine deacylation and demethylation critical for epigenetic and other cellular phenomena, using intramolecular O- to N-ester transfer reactions.     

Fluorescent small organic probes for biosensing

Small-molecule based fluorescent probes are increasingly important for the detection and imaging of biological signaling molecules due to their simplicity, high selectivity and sensitivity, whilst being non-invasive, and suitable for real-time analysis of living systems. With this perspective we highlight sensing mechanisms including Förster resonance energy transfer (FRET), intramolecular charge transfer (ICT), photoinduced electron transfer (PeT), excited state intramolecular proton transfer (ESIPT), aggregation induced emission (AIE) and multiple modality fluorescence approaches including dual/triple sensing mechanisms (DSM or TSM). Throughout the perspective we highlight the remaining challenges and suggest potential directions for development towards improved small-molecule fluorescent probes suitable for biosensing.

Small-molecule based fluorescent probes are increasingly important for the detection and imaging of biological signaling molecules due to their simplicity, high selectivity and sensitivity, whilst being non-invasive, and suitable for real-time analysis of living systems.     

A fluorescent molecular imaging probe with selectivity for soluble tau aggregated protein

Soluble forms of aggregated tau misfolded protein, generally termed oligomers, are considered to be the most toxic species of the different assembly states that are the pathological components of neurodegenerative disorders. Therefore, a critical biomedical need exists for imaging probes that can identify and quantify them. We have designed and synthesized a novel fluorescent probe, pTP-TFE for which binding and selectivity profiles towards aggregated tau and Aβ proteins were assessed. Our results have shown pTP-TFE to be selective for early forms of soluble tau aggregates, with high affinity of dissociation constants (K_d) = 66 nM, and tenfold selectivity over mature tau fibrils. Furthermore, we found that pTP-TFE is selective for tau over Aβ aggregates and had good cell permeability. This selectivity of pTP-TFE towards early forms of aggregated tau protein ex vivo was also supported with studies on human brain tissue containing tau and Aβ pathology. To the best of our knowledge, this is the first fluorescent molecule to be reported to have this form of selectivity profile, which suggests that pTP-TFE is a unique probe candidate for imaging-based detection of early stages of Alzheimer''s disease and other tauopathies.

pTP-TFE imaging probe can distinguish soluble tau aggregated proteins from other aggregated proteins enabling earlier detection of neurodegenerative diseases.     

In situ visualization of peroxisomal viscosity in the liver of mice with non-alcoholic fatty liver disease by near-infrared fluorescence and photoacoustic imaging

Non-alcoholic fatty liver disease (NAFLD) can gradually develop into hepatic failure, and early diagnosis is crucial to improve treatment efficiency. The occurrence of NAFLD is closely related to lipid metabolism. Peroxisomes act as the first and main site for lipid metabolism in the hepatocytes, so abnormal lipid metabolism might directly affect peroxisomal viscosity. Herein, we developed a new near-infrared fluorescence (NIRF) and photoacoustic (PA) imaging probe (PV-1) for the real-time visualization of peroxisomal viscosity in vivo. This PV-1 encompasses the malononitrile group as the rotor, which emits strong NIRF (at 705 nm) and PA (at 680 nm) signals when rotation is hindered as viscosity increases. Through dual-mode imaging, we discovered distinctly higher viscosity in the liver of NAFLD mice for the first time. We further found the remarkable amelioration of NAFLD upon treatment with N-acetylcysteine (NAC). Therefore, we anticipate that the PV-1 imaging method is promising for the early diagnosis and prognostic evaluation of NAFLD.

We report a novel near-infrared fluorescence/photoacoustic imaging method for peroxisomal viscosity, enabling an accurate diagnosis and drug evaluation of non-alcoholic fatty liver disease.     

Chemoenzymatic elaboration of the Raper–Mason pathway unravels the structural diversity within eumelanin pigments

Melanin is a central polymer in living organisms, yet our understanding of its molecular structure remains unresolved. Here, we apply a biosynthetic approach to explore the composite structures accessible in one type of melanin, eumelanin. Using a combination of solid-state NMR, dynamic nuclear polarization, and electron microscopy, we reveal how a variety of monomers are enzymatically polymerized into their corresponding eumelanin pigments. We demonstrate how this approach can be used to unite structure with an understanding of enzymatic activity, substrate scope, and the regulation of nanostructural features. Overall, this data reveals how intermediate metabolites of the Raper–Mason metabolic pathway contribute to polymerization, allowing us to revisit the original proposal of how eumelanin is biosynthesized.

Melanin is a central polymer in living organisms, yet our understanding of its molecular structure remains unresolved.     

Synthetic biohybrid peptidoglycan oligomers enable pan-bacteria-specific labeling and imaging: in vitro and in vivo

Peptidoglycan is the core component of the bacterial cell wall, which makes it an attractive target for the development of bacterial targeting agents. Intercepting its enzymatic assembly with synthetic substrates allows for labeling and engineering of live bacterial cells. Over the past two decades, small-molecule-based labeling agents, such as antibiotics, d-amino acids or monosaccharides have been developed for probing biological processes in bacteria. Herein, peptidoglycan oligomers, substrates for transglycosylation, are prepared for the first time using a top-down approach, which starts from chitosan as a cheap feedstock. A high efficiency of labeling has been observed in all bacterial strains tested using micromolar substrates. In contrast, uptake into mammalian cells was barely observable. Additional mechanistic studies support a hypothesis of bacteria-specific metabolic labeling rather than non-specific binding to the bacterial surface. Eventually, its practicality in bacterial targeting capability is demonstrated in resistant strain detection and in vivo infection models.

Peptidoglycan oligomers have been derived from chitosan, using a top-down bio-hybrid strategy, as highly bacteria-specific substrates.