Visualize intracellular ß-galactosidase using an asymmetric near-infrared fluorescent probe with a large Stokes shift.

ß-galactosidase (ß-Gal) is an important biomarker of cell senescence and primary ovarian cancer. Therefore, it is of great significance to construct a near-infrared fluorescent probe with deep tissue penetration and a high signal-to-noise ratio for visualization of ß-galactosidase in biological systems. However, most near-infrared probes tend to have small Stokes shifts and low signal-to-noise ratios due to crosstalk between excitation and emission spectra. Using d-galactose residues as specific recognition units and near-infrared dye TJ730 as fluorophores, a near-infrared fluorescence probe SN-CR with asymmetric structure was developed for the detection of ß-Gal. The probe has a fast reaction equilibrium time (<12 min) with ß-Gal, excellent biocompatibility, near-infrared emission (738 nm), low detection limit (0.0029 U/mL), and no crosstalk between the excitation spectrum and emission spectrum (Stokes shifts 142 nm) of the probe. Cell imaging studies have shown that SN-CR can visually trace ß-Gal in different cells and distinguish ovarian cancer cells from other cells.

Multiplexed assessment of engineered bacterial constructs for intracellular ß-galactosidase expression by redox amplification on catechol-chitosan modified nanoporous gold.

Synthetic biology approaches for rewiring of bacterial constructs to express particular intracellular factors upon induction with the target analyte are emerging as sensing paradigms for applications in environmental and in vivo monitoring. To aid in the design and optimization of bacterial constructs for sensing analytes, there is a need for lysis-free intracellular detection modalities that monitor the signal level and kinetics of expressed factors within different modified bacteria in a multiplexed manner, without requiring cumbersome surface immobilization. Herein, an electrochemical detection system on nanoporous gold that is electrofabricated with a biomaterial redox capacitor is presented for quantifying ß-galactosidase expressed inside modified Escherichia coli constructs upon induction with dopamine. This nanostructure-mediated redox amplification approach on a microfluidic platform allows for multiplexed assessment of the expressed intracellular factors from different bacterial constructs suspended in distinct microchannels, with no need for cell lysis or immobilization. Since redox mediators present over the entire depth of the microchannel can interact with the electrode and with the E. coli construct in each channel, the platform exhibits high sensitivity and enables multiplexing. We envision its application in assessing synthetic biology-based approaches for comparing specificity, sensitivity, and signal response time upon induction with target analytes of interest.

The ß-galactosidase assay in perspective: Critical thoughts for biosensor development.

Recently, the ß-galactosidase assay has become a key component in the development of assays and biosensors for the detection of enterobacteria and E. coli in water quality monitoring. The assay has often performed below its maximum potential, mainly due to a poor choice of conditions. In this study we establish a set of optimal conditions and provide a rough estimate of how departure from optimal values reduces the output of the assay potentially decreasing its sensitivity. We have established that maximum response for detecting low cell concentrations requires an induction of the samples using IPTG at a concentration of 0.2 mM during 180 min. Permeabilization of the samples is mandatory as lack of it results in an almost 60% reduction in assay output. The choice of enzyme substrate is critical as different substrates yield products with different extinction coefficients or fluorescence yields. The concentration of substrate used must be high enough (around 3 to 4 times Km) to ensure that the activity measured is not substrate limited. Finally, as the color/fluorescence of the reaction products is highly dependent on pH, care must be taken to ensure that pH at the time of reading is high enough to provide maximum signal.

Methods to Study Myc-Regulated Cellular Senescence: An Update.

Cellular senescence plays a role in several physiological processes including aging, embryonic development, tissue remodeling, and wound healing and is considered one of the main barriers against tumor development. Studies of normal and tumor cells both in culture and in vivo suggest that MYC plays an important role in regulating senescence, thereby contributing to tumor development. We have previously described different common methods to measure senescence in cell cultures and in tissues. Unfortunately, there is no unique marker that unambiguously defines a senescent state, and it is therefore necessary to combine measurements of several different markers in order to assure the correct identification of senescent cells. Here we describe protocols for simultaneous detection of multiple senescence markers in situ, a quantitative fluorogenic method to measure senescence-associated ß-galactosidase activity (SA-ß-gal), and a new method to detect senescent cells based on the Sudan Black B (SBB) analogue GL13, which is applicable to formalin-fixed paraffin-embedded tissues. The application of these methods in various systems will hopefully shed further light on the role of MYC in regulation of senescence, and how that impacts normal physiological processes as well as diseases and in particular cancer development.

Detecting Blue Light-Dependent Protein-Protein Interactions by LexA-Based Yeast Two-Hybrid Assay.

The LexA-based yeast two-hybrid system is one of the most powerful techniques used to detect blue light-dependent protein-protein interactions. In Arabidopsis, many protein-protein interactions in blue light signaling pathway were identified using this system. Here we present an easy and efficient method of the LexA-based yeast two-hybrid assay for testing protein-protein interactions in a blue light-dependent manner.

Chromo-fluorogenic probes for ß-galactosidase detection.

ß-Galactosidase (ß-Gal) is a widely used enzyme as a reporter gene in the field of molecular biology which hydrolyzes the ß-galactosides into monosaccharides. ß-Gal is an essential enzyme in humans and its deficiency or its overexpression results in several rare diseases. Cellular senescence is probably one of the most relevant physiological disorders that involve ß-Gal enzyme. In this review, we assess the progress made to date in the design of molecular-based probes for the detection of ß-Gal both in vitro and in vivo. Most of the reported molecular probes for the detection of ß-Gal consist of a galactopyranoside residue attached to a signalling unit through glycosidic bonds. The ß-Gal-induced hydrolysis of the glycosidic bonds released the signalling unit with remarkable changes in color and/or emission. Additional examples based on other approaches are also described. The wide applicability of these probes for the rapid and in situ detection of de-regulation ß-Gal-related diseases has boosted the research in this fertile field.

A sensitive fluorescent probe for ß-galactosidase activity detection and application in ovarian tumor imaging.

The development of non-invasive and sensitive optical probes for in vivo bioimaging of cancer-related enzymes is desirable for early diagnosis and effective cancer therapy. ß-galactosidase (ß-gal) is regarded as a key ovarian cancer biomarker, owing to its overexpression in primary ovarian cancer. Herein, we designed a sensitive near-infrared (NIR) probe (DCMCA-ßgal) for the detection and real-time imaging of ß-gal activity in ovarian tumors, thereby achieving the visualization of ovarian tumors by ß-gal activity detection. DCMCA-ß-gal could be triggered by ß-gal, resulting in the release of a NIR chromophore, DCM-NH2; the linear range of fluorescent response to ß-gal concentration was 0-1.2 U with a low detection limit of 1.26 × 10-3 U mL-1. We used DCMCA-ß-gal to detect and visualize ß-gal activity in SKOV3 human ovarian cancer cells, as well as for real-time imaging of ß-gal activity in ovarian cancer mouse models. DCMCA-ß-gal possessed high sensitivity, "turn-on" NIR emission, a large spectral shift, and high photostability in a dynamic living system and thus could serve as a highly sensitive sensor for real-time tracking of ß-gal activity in vivo and ovarian tumor imaging.

Far-red imaging of ß-galactosidase through a phospha-fluorescein.

The introduction of phosphine oxide into a fluorescein scaffold has yielded phospha-fluorescein with bathochromically shifted spectra, reliable photostability and solubility. Moreover, ratiometric and turn-on fluorescence in the decaging process has ensured that the phospha-fluorescein is a unique scaffold for fluorescence bioimaging. Probe DiMe-PF-Gal without further structural decoration was designed for accurately monitoring ß-galactosidase in vivo.

Photochromic Fluorescent Probe Strategy for the Super-resolution Imaging of Biologically Important Biomarkers.

Here, we report a ß-galactosidase (ß-Gal)-responsive photochromic fluorescent probe, NpG, that was designed to prebind to human serum albumin (HSA) to form the probe/protein hybrid, NpG@HSA. The formation of NpG@HSA led to an increase in fluorescence emission (520 nm) corresponding to the binding of the fluorescent naphthalimide unit with HSA. In addition, this enabled visualization of the spiropyran fluorescence emission in aqueous media. Our probe/protein hybrid approach afforded a unique imaging platform with enhanced cell permeability and solubility that was capable of visualizing the cellular uptake of NpG@HSA before its activation by ß-Gal. The ß-Gal-mediated cleavage of the galactose unit within the NpG@HSA hybrid resulted in the formation of NpM@HSA and an increase in red fluorescence emission (620 nm). The resultant merocyanine unit was then able to undergo photoisomerization (merocyanine ↔ spiropyran) to facilitate STORM (i.e., stochastic optical reconstruction microscopy) imaging with minimal phototoxicity and excellent photostability/reversibility. Using STORM, NpG@HSA was able to determine the subcellular distribution of ß-Gal activity between cell lines with nanoscale precision. We believe that this system represents a versatile imaging platform for the design of photochromic fluorescent probes suitable for illuminating the precise location of disease-specific biomarkers in various cellular processes.

In Vivo Imaging of Senescent Vascular Cells in Atherosclerotic Mice Using a ß-Galactosidase-Activatable Nanoprobe.

Senescence-associated diseases have severely diminished the quality of life and health of patients. However, a sensitive assay of these diseases remains limited due to a lack of straightforward methods. Considering that senescence-associated ß-galactosidase (SA-ß-Gal) is overexpressed in senescent cells, the detection of SA-ß-Gal in senescent cells and tissues might be a feasible strategy for the early diagnosis of SA diseases. In this study, a ß-galactosidase-activatable nanoprobe BOD-L-ßGal-NPs was developed for the imaging of senescent cells and vasculature in atherosclerotic mice via real-time monitoring of ß-Gal. BOD-L-ßGal-NPs was fabricated by encapsulating a newly designed NIR ratiometric probe BOD-L-ßGal within a poly(lactic-co-glycolic) acid (PLGA) core. Nanoprobe BOD-L-ßGal-NPs showed good accumulation in arteries, thus successfully visualizing senescent cells and vasculature in atherosclerotic mice by tail vein injection. Our findings indicated that nanoprobe BOD-L-ßGal-NPs holds great potential for the early diagnosis and therapy of atherosclerosis and other aging-associated diseases.

Multimode detection of ß-glycosidase and pathogenic bacteria via cation exchange assisted signal amplification.

A rapid strategy for the ß-glycosidase (ß-Gal) and Escherichia coli (E. coli) sensing is presented, which is based on selective recognition reactions of QDs using visualization/fluorescence (FL)/atomic fluorescence spectrometry (AFS)/inductively coupled plasma mass spectrometry (ICP-MS) multimode assay. CdTe QDs can selectively recognize Ag+ and Ag NPs with a cation exchange reaction (CER) where Ag+ triggers the release of Cd2+ and quenches the fluorescence signal of QDs. Taking advantage of the fact that ß-Gal can hydrolyze 4-Aminophenyl ß-D-galactopyranoside (PAPG) to produce p-aminophenol (PAP), which has the ability to reduce Ag+ to form Ag NPs. The ß-Gal can be easily detected by visualization or FL in a turn-on manner. Furthermore, combining with the selective separation of Cd2+ by filter membrane, AFS and ICP-MS with higher sensitivity were used for the determination of the enzyme. Under optimized conditions, the system limits of detections (LODs) were 0.01 U/L, 0.03 mU/L, and 0.02 mU/L using FL, AFS, and ICP-MS as the detector, respectively. The relative standard deviations (RSDs, n = 7) for 0.1 U/L ß-Gal were 2.2, 2.0, and 1.3% using FL/AFS/ICP-MS as the detector, respectively. And 0.1 U/L of ß-Gal can be discriminated from the blank solution with the naked eye. In addition, given that the ß-Gal can serve as an indicator of E. coli, we have successfully applied this strategy for the detection of E. coli with a LOD of 25 CFU/mL. Application of the method was demonstrated by analyzing human urine samples and milk samples for ultra-trace detection of E. coli. Graphical abstract The CVG-AFS/ICP-MS/visual/FL multimode ß-Gal and E.coli detection via CER.

No effect of the endurance training status on senescence despite reduced inflammation in skeletal muscle of older individuals.

The aim of the present study was to determine if the training status decreases inflammation, slows down senescence, and preserves telomere health in skeletal muscle in older compared with younger subjects, with a specific focus on satellite cells. Analyses were conducted on skeletal muscle and cultured satellite cells from vastus lateralis biopsies (n = 34) of male volunteers divided into four groups: young sedentary (YS), young trained cyclists (YT), old sedentary (OS), and old trained cyclists (OT). The senescence state and inflammatory profile were evaluated by telomere dysfunction-induced foci (TIF) quantification, senescence-associated ß-galactosidase (SA-ß-Gal) staining, and quantitative (q)RT-PCR. Independently of the endurance training status, TIF levels (+35%, P < 0.001) and the percentage of SA-ß-Gal-positive cells (+30%, P < 0.05) were higher in cultured satellite cells of older compared with younger subjects. p16 (4- to 5-fold) and p21 (2-fold) mRNA levels in skeletal muscle were higher with age but unchanged by the training status. Aging induced higher CD68 mRNA levels in human skeletal muscle (+102%, P = 0.009). Independently of age, both trained groups had lower IL-8 mRNA levels (-70%, P = 0.011) and tended to have lower TNF-α mRNA levels (-40%, P = 0.10) compared with the sedentary subjects. All together, we found that the endurance training status did not slow down senescence in skeletal muscle and satellite cells in older compared with younger subjects despite reduced inflammation in skeletal muscle. These findings highlight that the link between senescence and inflammation can be disrupted in skeletal muscle.

Visualization of endogenous ß-galactosidase activity in living cells and zebrafish with a turn-on near-infrared fluorescent probe.

ß-Galactosidase (ß-gal) is an important biomarker for primary ovarian cancers. Developing noninvasive bioimaging probes for studying the activity of ß-gal is highly desirable for cancer diagnosis. Herein, a turn-on near-infrared (NIR) fluorescent probe， 2-((6-(((2S, 3R, 4S, 5R, 6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran -2-yl)oxy)-2,3-dihydro-1H-xanthen-4-yl)methylene)malononitrile named DXM-ßgal, was rationally designed based on enzymatic reaction for the detection of ß-gal activity both in vitro and in vivo. Upon incubating with ß-gal, DXM-ßgal displayed a significant fluorescence enhancement at 640 nm, accompanying by a color change of solution color from red to purple. DXM-ßgal exhibited high selectivity and sensitively to ß-gal with low limit of detection (2.92 × 10-4 U mL-1). Besides, based on its advantages of long-wavelength emission and excellent biocompatibility, DXM-ßgal was successfully applied to imaging ß-gal in living cells and zebrafish. Given these prominent properties, we believe that DXM-ßgal will be a potential tool for investigating ß-gal activity in biomedical research.

Novel fluorescent probe for the ratiometric detection of ß-galactosidase and its application in fruit.

A ratiometric fluorescent probe (Probe 1) was developed for the sensitive detection of ß-galactosidase (ß-gal) activity. Probe 1 detected ß-gal activity in the range 0-1.0 U/mL, with a limit of detection of 0.025 U/mL. In addition, as different activities of ß-gal added, the luminescent intensity of Probe 1 gradually increased, as observed under a 365 nm ultraviolet lamp. Moreover, this method is low-volume, 20 µL, and time-efficient, 45 min per measurement. Probe 1 was successfully used to measure the ß-gal activity in real fruit samples in a qualitative manner, by the naked eye, fast semi-quantitative manner, by smartphone, or quantitative manner, by fluorescence spectrometer.

Contribution of senescence in human endometrial stromal cells during proliferative phase to embryo receptivity†.

Successful assisted reproductive technology pregnancy depends on the viability of embryos and endometrial receptivity. However, the literature has neglected effects of the endometrial environment during the proliferative phase on implantation success or failure. Human endometrial stromal cells (hESCs) were isolated from endometrial tissues sampled at oocyte retrieval during the proliferative phase from women undergoing infertility treatment. Primary hESC cultures were used to investigate the relationship between stemness and senescence induction in this population and embryo receptivity. Patients were classified as receptive or non-receptive based on their pregnancy diagnosis after embryo transfer. Biomarkers of cellular senescence and somatic stem cells were compared between each sample. hESCs from non-receptive patients exhibited significantly higher (P < 0.01) proportions of senescent cells, mRNA expressions of CDKN2A and CDKN1A transcripts (P < 0.01), and expressions of genes encoding the senescence-associated secretory phenotype (P < 0.05). hESCs from receptive patients had significantly higher (P < 0.01) mRNA expressions of ABCG2 and ALDH1A1 transcripts. Our findings suggest that stemness is inversely associated with senescence induction in hESCs and, by extension, that implantation failure in infertility treatment may be attributable to a combination of senescence promotion and disruption of this maintenance function in this population during the proliferative phase of the menstrual cycle. This is a promising step towards potentially improving the embryo receptivity of endometrium. The specific mechanism by which implantation failure is prefigured by a loss of stemness among endometrial stem cells, and cellular senescence induction among hESCs, should be elucidated in detail in the future.

Carbon dots-inspired fluorescent cyclodextrins: competitive supramolecular "off-on" (bio)sensors.

Chromophore-appended cyclodextrins combine the supramolecular loading capabilities of cyclodextrins (CDs) with the optical properties of the affixed chromophores. Among fluorescent materials, carbon dots (CNDs) are attractive and the feasibility of CND-appended CDs as sensors has been demonstrated by different authors. However, CNDs are intrinsically heterogeneous materials and their ulterior functionalization yields hybrid composites that are not well defined in terms of structure and composition. Inspired by the fluorescence properties of 5-oxo-1,2,3,5-tetrahydroimidazo[1,2-a]pyridine-7-carboxylic acid (IPCA), the most paradigmatic of the molecular fluorophores detected in CNDs, herein we report two highly efficient synthetic chemical strategies for the preparation of IPCA-appended CDs that behave as CND-based CD "turn off-on" biosensors suitable for the analysis of cholesterol and ß-galactosidase activity. We have deconstructed the CND-CD systems to demonstrate that (i) the role of CNDs is limited to acting as a support for the molecular fluorophores produced during their synthesis and (ii) the molecular fluorophores suffice for the determination of the enzymatic activity based on the quenching by p-nitrophenol as a sacrificial quencher.

Specific Near-Infrared Probe for Ultrafast Imaging of Lysosomal ß-Galactosidase in Ovarian Cancer Cells.

Reactivity based fluorescent probes have been widely investigated as a powerful and noninvasive tool for disease diagnosis in recent years. ß-Galactosidase (ß-gal), one of the typical lysosomal glycosidases, is reported to be a vital biomarker overexpressed in primary ovarian cancer cells. Fluorescent probes with excellent performance for endogenous ß-gal detection offer a unique option for visualization and diagnosis of primary ovarian cancer cells. Herein, a near-infrared fluorescent probe Lyso-Gal with lysosome-targeting ability was developed for lysosomal ß-gal detection and imaging in ovarian cancer cells (SKOV-3 cells). Lyso-Gal exhibits weak fluorescence in aqueous solution but emits bright NIR fluorescence at 725 nm after incubation with ß-gal. Highly selective imaging of ovarian cancer cells has been achieved upon incubation with Lyso-Gal for only 1 min. The detection time is extremely short. In comparison with a similar hemicyanine probe, Hx-Gal, without lysosome-targeting ability, Lyso-Gal realizes endogenous ß-gal visualization in lysosomes and shows brighter fluorescence than Hx-Gal in SKOV-3 cells. This work demonstrates the potential of Lyso-Gal for detection of primary ovarian cancer cells by using ß-gal as the biomarker.

Activatable Formation of Emissive Excimers for Highly Selective Detection of ß-Galactosidase.

Small-molecular fluorescence sensors have become promising detection tools in many fields attributing to their high sensitivity, excellent temporal and spatial resolution, and low cytotoxicity. However, high concentration or aggregation-induced fluorescence quenching effect has usually hindered the development of traditional fluorescence dyes. Herein, a new fluorophore cyanovinylene dye BMZ with excimer emission property has been constructed. It shows an obvious enhanced and red-shift emission upon aggregation in aqueous solution, which overmatches the conventional pyrene with longer absorption and emission wavelengths. Using this unique optical property, a new fluorescence probe BMZ-Gal has been developed for trapping of ß-galactosidase (ß-Gal) activity with high selectivity, low limit of detection of 0.17 U, and rapid recognition, which is based on the ß-Gal-induced formation of red-shift emitting excimer. ß-Gal has a strong affinity for BMZ-Gal, which is verified through the Michaelis-Menten constants (Km, 1.87 µM) and the hydrolysis efficiencies (Kcat/Km, 1.78 × 103 M-1 s-1). Furthermore, the assay system has been successfully used for detecting endogenous ß-Gal in living ovarian cancer cells and can passively targeted to identify ß-Gal in organelle level and determine its subcellular location with satisfactory accuracy. We anticipate that the new fluorophore cyanovinylene dye herein may inaugurate new opportunities for the development of excimer emission sensors.

Static microdroplet array generated by spraying and analyzed with automated microscopy and image processing.

Microdroplets have received increasing interest as practical platforms for high-throughput biochemical analysis. Typically, numerous discrete aqueous microdroplets containing biochemical targets are generated in a continuous oil phase and characterized using a flow-through configuration. Although this approach is capable of extremely high throughput, it is challenging to provide dynamic characterization of time-dependent reaction kinetics. In this paper, we present a practical and affordable method to create and analyze a massive array of static aqueous microdroplets immersed in oil for biochemical analysis. The discrete microdroplets were produced by an air-spray gun, imaged by automated microscopy, and then characterized by image processing. The location, area, and fluorescence intensity of randomly generated individual microdroplets were automatically registered for high-throughput characterization. With this approach, we rapidly produced and characterized a static microdroplet array of over 0.7 million microdroplets with an average volume of 300 fL and a mean population density of 1.5*105 microdroplets/cm2. Using the developed setup, we demonstrated the dependency of the microdroplets' fluorescence intensity on their volume, as well as characterized the time-dependent enzyme reaction kinetics of ß-galactosidase-mediated cleavage of the substrate fluorescein di-ß-d-galactopyranoside (FDG). The new approach described herein provides an inexpensive alternative solution for high-throughput analysis of dynamic biochemical processes.

In vivo ratiometric tracking of endogenous ß-galactosidase activity using an activatable near-infrared fluorescent probe.

Herein, we developed a dual-channel and light-up near-infrared fluorescent probe for ratiometric sensing of ß-galactosidase (ß-gal) activity. The well-designed probe, which shows ratiometric optical response with a significant red-shift (from 575 nm to 730 nm), was successfully applied to detect endogenous ß-gal activity in SKOV-3 cells and tumor-bearing mice.