Detection Of Β-galactosidase Research Articles

A novel NIR fluorescent probe for enhanced β-galactosidase detection and tumor imaging in ovarian cancer models

The advancement of biological imaging techniques critically depends on the development of novel near-infrared (NIR) fluorescent probes. In this study, we introduce a designed NIR fluorescent probe, NRO-βgal, which exhibits a unique off–on response mechanism to β-galactosidase (β-gal). Emitting a fluorescence peak at a wavelength of 670 nm, NRO-βgal showcases a significant Stokes shift of 85 nm, which is indicative of its efficient energy transfer and minimized background interference. The probe achieves a remarkably low in vitro detection limit of 0.2 U/L and demonstrates a rapid response within 10 min, thereby underscoring its exceptional sensitivity, selectivity, and operational swiftness. Such superior analytical performance broadens the horizon for its application in intricate biological imaging studies. To validate the practical utility of NRO-βgal in bio-imaging, we employed ovarian cancer cell and mouse models, where the probe's efficacy in accurately delineating tumor cells was examined. The results affirm NRO-βgal's capability to provide sharp, high-contrast images of tumor regions, thereby significantly enhancing the precision of surgical tumor resection. Furthermore, the probe's potential for real-time monitoring of enzymatic activity in living tissues underscores its utility as a powerful tool for diagnostics in oncology and beyond.

Design and synthesis of Lanthanide(III) complex based luminescence probes for time-resolved luminescence detection of β-galactosidase

β-Galactosidase (β-Gal), confirmed to provide energy and participate in the galactosyl transfer reaction, is primarily over-expressed in ovarian cancer cells. Herein, detecting endogenous β-Gal is essential for the diagnosis and monitoring of primary ovarian cancer cells. In this study, we proposed two structurally related cationic Lanthanide(III) triamide complexes for highly sensitive and rapid detection of β-Gal. Moreover, LDEu02 showed a low detection limit (2.3 × 10−3 U/mL) and high specificity with high affinity for β-Gal, which had great potential to the detection of the β-Gal activity by using time-resolved fluorescence techniques.

A double-enzyme cascade-responsive nanohydrogel for activatable photodynamic antibacterial therapy and dual fluorescence signal detection of bacterial infections

The tremendous morbidity and mortality caused by bacterial infections have emerged as a formidable menace to human health. The early detection and treatment of bacterial infections is imperative for effectively managing the progression of infectious diseases and mitigating infection-related mortality. In this study, we constructed a nanohydrogel consisting of cationic zinc phthalocyanine (CZP) and hyaluronic acid, which was further functionalized with a novel β-galactosidase detection probe, resulting in the formation of product CZP@HA:probe. The release of CZP and probe from CZP@HA:probe can be triggered by hyaluronidase, and the fluorescence of CZP and probe was turned on successively by hyaluronidase and β-galactosidase, respectively, demonstrating the dual enzyme-responsive property of CZP@HA:probe. CZP@HA:probe exhibited potent photodynamic killing activity against bacteria. The antibacterial mechanism potentially involved the generation of reactive oxygen species within bacteria and the disruption of the bacterial outer membrane. CZP@HA:probe had significant efficacy in promoting wound healing and exhibited a fluorescent indication of wound infections. The construction strategy of the nanohydrogel can be applied to other photosensitizers and probes, offering a novel perspective for the detection and treatment of bacterial infections.

A near-infrared fluorescent probe with a substantial Stokes shift designed for the detection and imaging of β-galactosidase within living cells and animals

β-Galactosidase serves as a pivotal biomarker for both cancer and cellular aging. The advancement of fluorescent sensors for tracking β-galactosidase activity is imperative in the realm of cancer diagnosis. We have designed a near-infrared fluorescent probe (PTA-gal) for the detection of β-galactosidase in living systems with large Stokes shifts. PTA-gal exhibits remarkable sensitivity and selectivity in detecting β-galactosidase, producing near-infrared fluorescent signals with a remarkably low detection limit (2.2 × 10-5 U/mL) and a high quantum yield (0.30). Moreover, PTA-gal demonstrates biocompatibility and can effectively detect β-galactosidase in cancer cells as well as within living animals.

Functionalized-Graphene Field Effect Transistor-Based Biosensor for Ultrasensitive and Label-Free Detection of β-Galactosidase Produced by Escherichia coli.

The detection of β-galactosidase (β-gal) activity produced by Escherichia coli (E. coli) can quickly analyze the pollution degree of seawater bodies in bathing and fishing grounds to avoid large-scale outbreaks of water pollution. Here, a functionalized biosensor based on graphene-based field effect transistor (GFET) modified with heat-denatured casein was developed for the ultrasensitive and label-free detection of the β-gal produced by E. coli in real water samples. The heat-denatured casein coated on the graphene surface, as a probe linker and blocker, plays an important role in fabricating GEFT biosensor. The GFET biosensor response to the β-gal produced by E. coli has a wide concentration dynamic range spanning nine orders of magnitude, in a concentration range of 1 fg·mL-1-100 ng·mL-1, with a limit of detection (LOD) 0.187 fg·mL-1 (1.61 aM). In addition to its attomole sensitivity, the GFET biosensor selectively recognized the β-gal in the water sample and showed good selectivity. Importantly, the detection process of the β-gal produced by E. coli can be completed by a straightforward one-step specific immune recognition reaction. These results demonstrated the usefulness of the approach, meeting environmental monitoring requirements for future use.

β-galactosidase activatable chemiluminescent probe for in vivo imaging and point-of care test

As a highly effective tool with a superior signal-to-noise ratio, chemiluminescent probes find extensive utility in the in vivo detection of diverse biochemical aspects, including inflammation and the tumor microenvironment. However, the widespread adoption of chemiluminescence detection is hindered by the requirement of large precision instruments, thereby limiting its application scope. Consequently, further development and improvement of the chemiluminescence detection method are imperative. This study presents a novel chemiluminescent probe that enables precise detection of β-galactosidase (β-gal) both in vitro and in vivo by modifying the specific cleavage recognition groups of β-gal to the hydroxyl group of the dioxane-based chemiluminescent scaffold. In comparative tests against traditional chemical probes, this probe exhibited an exceptional signal-to-background ratio that significantly surpasses its counterparts. To enhance detection capabilities, a customized Point-of-Care Testing (POCT) instrument specifically designed for chemiluminescence detection was developed. This compact, user-friendly, and cost-effective instrument demonstrated comparable performance to large precision instruments. Notably, the structural characteristics of the probe, utilizing the 'hydroxyl deprotection' strategy, allow for convenient, real-time, and accurate detection of diverse targets by modifying the recognition groups tailored to specific targets, in conjunction with the POCT instrument. This research introduces a pioneering approach for generating optical signal tests in the laboratory using chemiluminescent probes, thereby unlocking the potential for rapid clinical detection and self-diagnosis of certain diseases in a home setting.

Ultrasensitive two-photon probes for rapid detection of β-galactosidase during fruit softening and cellular senescence

Senescence is happening in every corner of the living organisms. β-galactosidase (β-gal) is one of the most important biomarkers during senescence in both plant and mammalian cells. Most β-gal fluorescent probes were focused on bio-imaging, only a few probes were developed for the detection of β-gal in fruit, and the probes that could detect β-gal in both fruits and living cells were even less. Here, two β-gal probes (TNap-βGal and TBNap-βGal) were synthesized, which can not only image the increase of β-gal during both fruits softening and cellular senescence, but also prove that bananas are not suitable for storage in refrigerator and the subsequent accumulation of β-gal still in lysosome of mammalian cells. In addition, TNap-βGal was successfully applied to two-photon imaging of endogenous β-gal in both hDPMSCs and tissues of human dental pulp for the first time.

Senescence-Associated β-Galactosidase Detection in Pathology.

Activity of β-galactosidase at pH 6 is a classic maker of senescence in cellular biology. Cellular senescence, a state of highly stable cell cycle arrest, is often compared to apoptosis as an intrinsic tumor suppression mechanism. It is also thought that SA-β-gal is crucial in malignant cell transformation. High levels of senescence-associated β-galactosidase (SA-β-gal) can be found in cancer and benign lesions of various localizations making the enzyme a highly promising diagnostic marker for visualization of tumor margins and metastases. These findings facilitate the research of therapy induced senescence as a promising therapeutic strategy. In this review, we address the need to collect and analyze the bulk of clinical and biological data on SA-β-gal mechanisms of action to support wider implementation of this enzyme in medical diagnostics. The review will be of interest to pathologists, biologists, and biotechnologists investigating cellular senescence for purposes of regenerative medicine and oncology.

Green-emission nitrogen-doped carbon quantum dots from alkaline N-methyl-2-pyrrolidinone for determination of β-galactosidase and its inhibitors.

Anew fluorescence method was established for sensitive detection of β-galactosidase (β-gal) activity in spiked human serum and screeningof inhibitor. Nitrogen-doped carbon quantum dots (N-CQDs) were prepared by solvothermal polymerization of N-methyl-2-pyrrolidinone in an alkaline condition. The colloidal N-CQDs exhibit good water solubility, stability, and emit bright green fluorescence with a maximum emission peak at 528nm upon excitation at 420nm. β-gal specifically catalyzes the decomposition of its substrate P-nitrophenyl-β-D-galactopyranoside into 4-nitrophenol, whose absorption spectrum overlaps well with the excitation spectrum of the N-CQDs. As a result, the fluorescence of the N-CQDs is remarkably quenched by 4-nitrophenol via an inner filter effect. The sensing platform presents a linear response range for β-gal activity from 0.05 to 3.0 U·L-1 with a low limit of detection of 0.023 U·L-1. An acceptable precision is obtained with a relative standard deviation (RSD) of 3.1% for 1.0 U·L-1 β-gal (n = 11). The method was applied to determine β-gal in spiked human serums with recoveries in the range 96.3-104.7%. The methodwas employed to evaluate inhibitor screening with D-galactal and chloroquine diphosphate as models.

Kill two birds with one stone: A near-infrared ratiometric fluorescent probe for simultaneous detection of β-galactosidase in senescent and cancer cells

β-galactosidase, as a typical lysosomal glycosidase, is one of the irreplaceable markers in ovarian cancer and senile cells. Therefore, a new method to detect endogenous β-gal is urgently needed to better understand its role in related biological processes. Here, a novel near-infrared proportional fluorescent probe was designed and screened to detect the activity of β-gal in cancer and senescent cells. The probe achieved a rapid response (within 15 min) to β-gal, a large stoke-shift (170 nm), and an 80-fold fluorescence enhancement with a maximum emission wavelength of 670 nm after interaction with β-gal. The probe has good biocompatibility and can effectively avoid the interference of biological background. Subsequently, the developed probe was used to detect and image endogenous β-gal in cells and zebrafish. In addition, TZ-Br has been successfully used to evaluate the therapeutic effects of dasatinib and quercetin combination on senile cells, which provides a powerful tool for further understanding the physiological and pathological functions of β-gal in living systems.

Puerarin promotes apoptosis and senescence of bladder cancer cells

• Puerarin inhibits the proliferation and induces apoptosis and senescence of bladder cancer cells. • Puerarin exerts anticancer effects by up-regulating miR-139-5p in bladder cancer cells. • CCNB1 is the target gene of miR-139-5p. • Puerarin down-regulates CCNB1 in bladder cancer cells. • Puerarin down-regulates CCNB1 and PI3K/AKT pathways through miR-139-5p. This study aims to explore the mechanism of puerarin regulating the proliferation, apoptosis and senescence of T24 and EJ cells via miR-139-5p/CCNB1 and PI3K/AKT pathways. Initially, Cell Counting Kit ( CCK)-8, flow cytometry, β-galactosidase detection kit, and RT-qPCR are used to assess the effect of puerarin on the proliferation apoptosis and senescence of bladder cancer cells by regulating miR-139-5p, respectively. Then, potential complementary binding sites between miR-139-5p and the 3′ untranslated region (3′ UTR) of the CCNB1 gene are obtained by the bioinformatics software, starBase database. Further, a dual-luciferase reporter system is used to assess the targeted relationship of miR-139-5p to CCNB1. CCK-8, flow cytometry, and β-galactosidase detection kit were used to detect the influence of CCNB1 on the proliferation, apoptosis, and senescence of bladder cancer cells. RT-qPCR and Western blotting are used to determine the regulation of CCNB1 and PI3K/AKT pathways by miR-139-5p. Finally, the subcutaneous tumor formation in nude mice and immunohistochemical experiments are performed to assess the effect of puerarin on the proliferation of bladder cancer cells in vivo . Puerarin induced proliferation inhibition, apoptosis, and senescence of bladder cancer cells in vitro . Puerarin down-regulated the expression of CCNB1, p-PI3K, and p-AKT proteins by overexpressing miR-139-5p, thereby exerting a tumor suppressor effect in bladder cancer. Compared with the control group, the volume and weight of subcutaneous tumors of nude mice in the puerarin administration group are reduced. Moreover, the ki-67 positive staining rate and the expression level of CCNB1, p-PI3K, and p-AKT protein are reduced. The experimental results showed that the anti-tumor effect of puerarin against bladder cancer might be achieved by inhibiting CCNB1 and PI3K/AKT pathway via up-regulating miR-139-5p.

A novel AIE fluorescent probe for β-galactosidase detection and imaging in living cells

An AIE fluorescent probe (TPh-PyBz-β-gal) was designed and synthesized for β-gal detection based on a novel AIE fluorophore and receptor unit D-galactose residue connected by glycosidic bond. The probe exhibited selective and sensitive turn-on fluorescent responses toward β-gal with detection limit reaching to 0.22 U/mL in aqueous environment. After studied from measured data, we proposed that a possible recognition mechanism probably resulted from the cleavage of glycosidic bond along with obviously increasing fluorescent signal at 606 nm. Meanwhile, TPh-PyBz-β-gal showed low cytotoxicity, and could be successfully used for β-gal imaging in cells.

Kill Two Birds with One Stone: A Near-Infrared Ratiometric Fluorescent Probe for Simultaneous Detection of Β-Galactosidase in Senescent and Cancer Cells

Kill Two Birds with One Stone: A Near-Infrared Ratiometric Fluorescent Probe for Simultaneous Detection of Β-Galactosidase in Senescent and Cancer Cells

A new near-infrared excitation/emission fluorescent probe for the detection of β-galactosidase in living cells and in vivo

Development of noninvasive bioimaging fluorescent probes for detecting particular enzyme activity is greatly recommendable for preclinical diagnosis of cancer. Given that the elevated β-gal activity is positively correlated with several tumors, developing a fluorescent probe for the sensing of β-gal is therefore highly desirable for cancer diagnosis. Herein, a new enzyme-activatable near-infrared (NIR) turn-on fluorescent probe (DMC-βgal) was developed for accurately detecting β-gal activity characterized by excellent selectivity, high sensitivity (LOD = 0.298 U/L), and low toxicity. More importantly, DMC-βgal qualifies remarkable NIR excitation (725 nm) and emission wavelength (770 nm), an ideal tool for restrained photodamage and suppressed autofluorescence. The above excellent performance of DMC-β-gal allowed for the accurate monitoring of endogenous β-gal in living cells. Moreover, the probe was successfully applied to detect intracellular β-gal activity in different types of cancer cells, verifying that SKOV-3 cells had a higher level of β-gal activity than those of A549, HCT-116, MCF-7, and HepG2 cells. Furthermore, DMC-βgal could real-time visualize endogenously β-gal in tumor-bearing nude mice with low auto-fluorescence interference. All results fully demonstrated that DMC-βgal has potential value as a promising strategy for diagnosis of β-gal-related diseases.

Nanoparticle-based amplification for sensitive detection of β-galactosidase activity in fruits

Development of fast and sensitive assays for enzyme activity detection has received a great deal of attention because of the wide spread applications in measurements of numerous clinical, food and environmental processes. Herein, a novel amplification approach to enhance the sensitivity of colorimetric assays for detection of β-galactosidase (β-Gal) activity is proposed. β-Gal detection is important in biomedical applications and in food industry, where it is associated with the ripening process of fruits. The method is based on the use of multivalent cerium oxide nanoparticles (CeNPs) which catalyze the oxidation of 4-aminophenol (4-AP) produced in the hydrolysis process of the 4-aminophenyl-β-d-galactopyranoside substrate (4-APG) by β-Gal, thus enhancing detection sensitivity of β-Gal in the visible range. The developed assay is highly sensitive and easy to use. Using the optimized procedure, a limit of detection of 0.06 mU/mL was obtained with a linearity range up to 2.0 mU/mL. The feasibility of the method was demonstrated for detection of β-Gal activity in fruits and the results were compared with the conventional assay, providing over a 30-fold amplification as compared to a commercially available β-Gal protocol. The advantage of the presented assay is its biocatalytic event amplified by a secondary reaction, which enables much more sensitive detection of the enzymatic product. The sensing platform can be applied broadly to a variety of applications that rely on β-Gal activity measurements.

First aggregation-induced emission-active probe for species-specific detection of β-galactosidase

Sensitive detection of β-galactosidase (β-gal) is of great significance for early diagnosis of ovarian cancer. Fluorescent probes for detecting β-gal have received great interest due to the non-invasiveness, excellent sensitivity, high temporal, and superior spatial resolution. However, most reported fluorescent sensors for β-gal suffer from aggregation caused quenching effect when accumulated, and cannot discriminate β-gal from other species, especially, Escherichia coliβ-gal. Herein, we report the first aggregation-induced emission (AIE)-active fluorescent probe HBTTPAG, which achieves species-selective detection of β-gal. Probe HBTTPAG can discriminate Aspergillus oryzae β-gal from Escherichia coliβ-gal, with high sensitivity (detection limit of 3.7 × 10-3 UmL-1), superior selectivity and low cytotoxicity. Furthermore, HBTTPAG is utilized to visualize endogenous β-gal in lysosomes of SKOV-3 cells, as well as to detect β-gal activity in ovarian cancer tissues. Notably, owing to the AIE-active, HBTTPAG realizes long-term (12 h) tracking β-gal in ovarian cancer cells. This work provides a promising method for species-selective detection of β-gal in preclinical.

New insights into the detection mechanism of β-galactosidase in living cells with fluorescent probes

The mechanism of β-galactosidase detection probes ESIPT-GAL and ESIPT-OH have been investigated. The torsional potential energy curve proves the planar characteristics of the system. Combined with frontier molecular orbital and “hole-electron” analysis, there are apparent planar intramolecular charge transfer (PICT) characteristics in ESIPT-GAL and ESIPT-OH. The barrier-free intramolecular proton transfer explained the single fluorescence. Furthermore, the mechanism of ESIPT-OH fluorescent probe is the cooperation of excited-state intramolecular proton transfer (ESIPT) and PICT. The relationship between the singlet and triplet states combined with the spin coupling constant reveals that the weak fluorescence characteristic of ESIPT-GAL is caused by the intersystem crossing.

A “turn-on” fluorescent probe for ultra-sensitive detection of β-galactosidase

The detection of β-galactosidase is significant for identifying the occurrence of ovarian cancer and cellular senescence. Since the complexity and diversity of biological systems, fluorescent probes for biological detection are required to own good selectivity, high selectivity and strong anti-interference ability. To meet this challenge, we designed a “turn-on” fluorescent probe TC-gal by adjusting the effect of twisted intramolecular charge transfer (TICT) between intramolecular charge transfer (ICT) of the fluorophore for ultra-sensitive detecting of β-galactosidase. The β-galactosidase can specifically trigger an enhanced (more than 800 folds) light-up fluorescence of TC-gal due to attenuated TICT effect of tetraphenylethylene part and increased ICT effect induced by electron donating effect of generated hydroxyl. Such “turn-on” fluorescence selectively triggered by β-galactosidase endows probe TC-gal with low background fluorescence and high signal-to-noise ratio. It is demonstrated that designed TC-gal exhibits high selectivity, ultra-high sensitivity (LOD = 8.4 × 10−5 U/mL) and strong affinity (Km = 18.4 μM) to β-galactosidase in buffer solution with good anti-interference ability to other biological analytes. Furthermore, biocompatible probe TC-gal is successfully applied to detect endogenous β-galactosidase in ovarian cancer cells and distinguish senescent cells. We anticipate this work can inspire the generation of superior fluorescent biosensors for biological and medical application.

Highly sensitive protein detection using recombinant spores and lateral flow immunoassay.

Lateral flow immunoassays (LFIs) can be used to detect intact bacteria or spores; when gold nanoparticles (AuNPs) are used as the signal reporters, the detection limits are very low. Spore-based surface display has been widely studied for enzyme immobilization and live-nontoxic oral vaccines. In this study, recombinant spores were used to improve the sensitivity of a LFI. We developed a test kit that combines streptavidin-displayed spores with a LFI assay for rapid protein detection. The recombinant spores served as a signal amplifier and AuNPs were used as the signal reporters. For detection of β-galactosidase, which was used as the model protein, the detection limit was about 10-15mol, while that of the conventional LFI is about 10-12mol. In both methods, nanogold was used as the colorimetric signal and could be observed with the naked eye. This method improved LFI sensitivity without sacrificing its advantages. Furthermore, enhanced green fluorescent protein (eGFP) was also displayed on the surface of the streptavidin-displayed spores. Without AuNPs, the fluorescent recombinant spores acted as the signal, which could be detected by a fluorescence detector, such as a fluorescence microscope. The detection limit was 10-16mol under fluorescence microscopy whose magnification was 25-fold. Therefore, in conclusion, in this proof of concept study, the detection limits of both proposed methods were far superior to those of traditional LFI assay.

Chromo-fluorogenic probes for β-galactosidase detection.

β-Galactosidase (β-Gal) is a widely used enzyme as a reporter gene in the field of molecular biology which hydrolyzes the β-galactosides into monosaccharides. β-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 β-Gal enzyme. In this review, we assess the progress made to date in the design of molecular-based probes for the detection of β-Gal both in vitro and in vivo. Most of the reported molecular probes for the detection of β-Gal consist of a galactopyranoside residue attached to a signalling unit through glycosidic bonds. The β-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 β-Gal-related diseases has boosted the research in this fertile field.