Search Results (77)

Phenol red is a widely used, low-cost, label-free colorimetric pH indicator that bridges traditional colorimetric assays with modern quantitative imaging and cell-based screening platforms. Its protonation-dependent absorbance shift (430–560 nm) allows for the real-time monitoring of extracellular acidification, which indirectly reflects cellular metabolism, growth, and respiration. Although phenol red lacks the molecular specificity of genetically encoded or fluorogenic biosensors, it remains useful in systems where pH changes are effective proxies for physiological processes. Existing tissue digestion protocols often overlook key parameters, especially pH control and enzyme cofactor use. This study presents a straightforward, spectrophotometric method to monitor and adjust the pH of low-volume (1 mL) buffered enzymatic dissociation media using phenol red and a plate reader. We titrated dissociation solutions to physiological pH (~7.4) using spectrophotometric pH measurements validated against conventional glass pH probe readings, confirming method reliability. Accurate pH assessment is critical for isolating viable primary cells for downstream applications such as tissue engineering, single-cell omics, and neurophysiological assays. We highlight that papain-based dissociation media supplemented with L-cysteine can be acidic (pH 6.6) if unadjusted, compromising cell viability. This accessible approach enhances reproducibility by promoting pH documentation concerning dissociation conditions that contribute to advancing consistency in biomedical, cellular, neuronal, and tissue engineering research. Full article

Tuberculosis remains a serious global public health challenge and requires the development of rapid, sensitive, and specific diagnostic tools for effective treatment and disease control. Bioimaging reporters are promising diagnostic tools that exploit the unique biochemical properties of Mycobacterium tuberculosis for real-time detection of viable cells from clinical samples. Moreover, these methods offer significant advantages over the conventional methods currently used in practice, including reduced assay time, increased specificity, and the ability to discriminate viable cells from dead cells. In this review, we highlight reporters of a different nature that the enable direct detection of Mycobacterium tuberculosis, eliminating complex sample preparation. Such reporters could serve as powerful tools in fluorescence microscopy, provide alternative strategies for automated culture-based diagnostic systems, and offer new approaches for developing point-of-care methods and diagnostic devices suitable for clinical practice. Full article

Using an environmentally friendly approach, we successfully synthesized an asymmetric monomethine cyanine dye, 7-chloro-1-ethyl-4-((3-ethylbenzo[d]thiazol-2(3H)-ylidene)methyl) quinolin-1-ium iodide, named CHLoris (CHL), via a modified Knoevenagel-type condensation. The reaction was carried out mechanochemically in an ethanol–water medium using 1-ethyl-2-methylbenzothiazolium iodide and 4,7-dichloro-1-ethylquinolin-1-ium iodide in the presence of sodium carbonate as a base and catalytic amounts of Hünig’s base. The UV/VIS absorption spectra of CHL in both the buffer solution and ethanol revealed the formation of aggregates in aqueous media. Density Functional Theory (DFT) and Time-Dependent DFT (TDDFT) calculations were employed to support the experimental findings further and provide insights into the self-association behavior of CHL in an aqueous solution. The photophysical properties of the dye were examined in the presence of DNA and RNA, and its performance was compared to that of the commercial dye Thiazole Orange (TO) under identical conditions. The results show that CHL is more sensitive towards RNA. Full article

The recent COVID-19 pandemic has made the public aware of the importance of combating pathogenic microorganisms before they enter the human body. This growing threat from microorganisms prompted us to conduct research into a new type of coating that would be an alternative to the continuous disinfection of touch surfaces. Our goal was to design, synthesise and thoroughly characterise such a coating. In this work, we present a nanocomposite material composed of a thin-layer mesoporous SBA-15 silica matrix containing copper phosphonate groups, which act as catalytic centres responsible for the generation of reactive oxygen species (ROS). In order to verify the structure of the material, including its molecular structure, microscopic observations and Raman spectroscopy were performed. The generation of ROS was confirmed by fluorescence microscopy analysis using a fluorogenic probe. The antimicrobial activity was tested against a wide spectrum of Gram-positive and Gram-negative bacteria, while cytotoxicity was tested on BALB/c3T3 mouse fibroblast cells and HeLa cells. The studies fully confirmed the expected structure of the obtained material, its antimicrobial activity, and the absence of cytotoxicity towards fibroblast cells. The results obtained confirmed the high application potential of the tested nanocomposite coating. Full article

Malondialdehyde (MDA), a mutagenic and carcinogenic compound, is widely studied in the meat industry and lipid peroxidation research due to its implications for food quality and safety. Current methods for quantifying MDA in solid tissues are labor-intensive, requiring multiple instruments and approximately two hours to complete. This study presents an ultrafast kinetic fluorogenic method for quantifying MDA in ground beef, utilizing 2-thiobarbituric acid (TBA) as a fluorogenic probe. The total assay time is significantly shortened to 6 min from sample preparation to data acquisition. The assay’s robustness against matrix interference was validated using sample volume variation and standard addition calibration methods. Additionally, the effects of ambient exposure to air, washing, and cooking on MDA content in raw ground beef were quantified. While both ambient exposure to air and cooking increased MDA levels, washing raw ground beef and decanting cooked ground beef broth effectively reduced MDA levels in the ground beef. This simple and rapid assay can be adopted both in food research and industry. Moreover, insights from our study on the relationship between ground beef treatment and MDA concentration will help consumers make informed decisions about ground beef handling and consumption to lower their intake of MDA. Full article

In this work, we propose an automated, real-time optical scanning approach to assessing catalyst performance in the process of nitro-to-amine reduction using well-plate readers to monitor reaction progress. This approach takes advantage of a simple on–off fluorescence probe that gives a shift in absorbance and strong fluorescent signal when the non-fluorescent nitro-moiety is reduced to the amine form. The combination of an affordable probe and a low barrier-to-entry technique provides an accessible approach to high-throughput catalyst screening. Under this paradigm, we screened 114 different catalysts and compared them in terms of reaction completion times, material abundance, price, recoverability, and safety. Using a simple scoring system, we plotted the catalysts in terms of cumulative scores, along with some intentional biases, including an emphasis on preference for catalysts with potential as green catalysts, considering environmental issues and possible geopolitical preferences. Full article

DNA phosphorothioate (PT) modifications, characterized by the replacement of a non-bridging phosphate oxygen atom with a sulfur atom, are widely observed in bacterial genomes. Sensitive detection of phosphorothioate is crucial for elucidating their biological roles and functions. Herein, we developed an innovative method that leverages oligonucleotide-templated reactions (OTRs) and fluorogenic oligonucleotide probes. By optimizing temperature, probe sequence length, and the relative distance between PT position and the fluorophore probe, we achieved sensitive detection for DNA PT modifications through fluorogenic signal amplification, which provides an efficient and cost-effective approach for sensitive detection of phosphorothioate-modified DNA. Full article

Insulin-like growth factor-1 (IGF-1) is a regulatory factor closely associated with diabetes, obesity, and breast cancer, and it also acts as one of the most abundant growth factors in bovine colostrum. Current methods generally have the problem of low sensitivity, a time-consuming nature, and low stability, which makes it difficult to crack down on the false advertising of IGF-1 content in dairy products. In this work, an ultrasensitive fluorescent enzyme-linked immunosorbent assay (ELISA) is proposed, where the antibody and the target are combined in the form of a “sandwich” to ensure the accuracy and specificity of the assay. IGF-1 is quantified based on an effective hydrogen peroxide (H₂O₂) probe with 10-acetyl-3,7-dihydroxyphenoxazine (ADHP) as the fluorogenic substrate. The proposed fluorescent sandwich ELISA has a low limit of detection (LOD) of 77.29 pg/mL, fast experimental process within 1 h, and stable signal of 1 h. Furthermore, multi-step pretreatment methods for bovine colostrum powders are established to remove interfering substances, including fat, casein, and binding proteins, achieving the accurate and specific detection of IGF-1. IGF-1 recovery studies on treated bovine colostrum powders exhibit good recovery rates ranging from 91.71% to 102.32%, which proves the feasibility of detecting IGF-1 in real bovine colostrum. Full article

Arsenic is a well-known, highly toxic carcinogen element that is widely found in nature, with numerous studies highlighting its hazardous impact on human health and the environment. Therefore, considering its toxicity and adverse health effects on mammals and the environment, rapid, sensitive, and effective methods for the recognition of arsenic are necessary. Over the past decade, a variety of fluorescent probes, such as small molecules, nanomaterials, gold nanoparticles (AuNPs), carbon dots (CDs), quantum dots (QDs), and more, have been designed and successfully employed for the recognition of lethal arsenic. Compared to other conventional sensor materials, sensors based on metal-organic frameworks (MOFs) are advantageous due to their simple preparation, easy functional group modulation, large specific surface area, and excellent chemical stability. In recent years, MOFs have been utilized as dual-functional materials for the detection and adsorptive removal of arsenic from water. This unique functionality distinguishes MOF-based materials from conventional sensors and arsenic adsorbents. Herein, we provide an overview of the state-of-the-art knowledge on the current development of MOFs for the fluorogenic detection of arsenic in aqueous media. Furthermore, the underlying detection mechanisms are also summarized in this review. The existing challenges in this field and potential remedial strategies for improving detection are elaborated upon in the relevant sections. Full article

Two new asymmetric monomethine cyanine dyes, featuring dimethoxy quinolinium or methyl quinolinium end groups and benzothiazole or methyl benzothiazole end groups were synthesized. The chemical structures of the two dyes—(E)-6,7-dimethoxy-1-methyl-4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium iodide (3a) and (E)-4-((3,5-dimethylbenzo[d]thiazol-2(3H)-ylidene)methyl)-1,2-dimethylquinolin-1-ium iodide (3b)—were confirmed through NMR spectroscopy and MALDI-TOF mass spectrometry. A new methodology was developed to study monocationic dyes in the absence of a matrix and cationizing compounds in MALDI-TOF mass experiments. The newly synthesized dyes contain hydrophobic functional groups attached to the chromophore, enhancing their affinity for the hydrophobic regions of nucleic acids within the biological matrix. The dyes’ photophysical properties were investigated in aqueous solutions and DMSO, as well as in the presence of nucleic acids. The dyes exhibit notable aggregachromism in both pure aqueous and buffered solutions. The observed aggregation phenomena were further elucidated using computational methods. Fluorescence titration experiments revealed that upon contact with nucleic acids, the dyes exhibit bioaggregachromism–aggregachromism on the surfaces of the respective biomolecular matrix (RNA or DNA). This bioaggregachromism was further confirmed by CD spectroscopy. Given the pronounced aggregachromism detected, we conclude that the dyes investigated in this study are highly suitable for use as fluorogenic probes in biomolecular recognition techniques. The unique absorption and fluorescence spectra of these dyes make them promising fluorogenic markers for various bioanalytical methods related to biomolecular recognition. Full article

As the last resort and one of the most crucial antibiotics for multidrug-resistant bacteria, carbapenem is considered the best hope for treating bacterial infections. However, the prompt emergence of carbapenemase-producing bacteria (CPB) poses a striking global health threat. Thus, accurate and rapid methods for the detection of carbapenemase are being requested to guide precise diagnosis, appropriate treatment strategies, and antibiotic stewardship. Although genotypic, phenotypic, and biochemical methods are currently used in clinical practice for CPB detection, they each have their problems that cannot commendably meet the need. In recent years, small-molecule probes have made significant progress and breakthroughs in the rapid detection and subtyping of CPB, providing insights and innovative solutions for the ultra-sensitive detection of CPB. In this minireview, some of the advances, namely, chromogenic probes and methods, fluorogenic probes, dual fluorogenic–chromogenic probes, a chemiluminescent probe, and a novel label-free intracellular calorimetric approach, are summarized, appreciated, and discussed. These methods offer high sensitivity, specificity, and accuracy in a short period in clinical settings without the utilization of sophisticated equipment or professional personnel. We hope that this minireview can provide a reference for the development of rapid detection of CPB and eventually contribute to antibiotic resistance management. Full article

The precise and fast detection of heparin, the most widely used anticoagulant, remains a significant challenge for assessing its use in a clinical setting. In this work, we adapt a well-established asymmetric cyanine fluorogenic platform for the purpose of ultrasensitive heparin detection in the presence of common interferant chemical species. Three analogous fluorescence probes are synthesized in order to optimize for the number of binding moieties. Their interaction with heparin is studied using steady-state absorption, fluorescence, and circular dichroism spectroscopy. The obtained probes exhibit a highly sensitive “turn-on” fluorescence response to heparin, with a LOD in the 10–25 nM range, well within practical requirement, as well as a visible colorimetric change. The heparin–probe complex is also employed as a sensitive detection platform for protamine, both in the “turn-off” fluorescence and ratiometric detection schemes. Full article

The development of fluorescence-based methods for bioassays and medical diagnostics requires the design and synthesis of specific markers to target biological microobjects. However, biomolecular recognition in real cellular systems is not always as selective as desired. A new concept for creating fluorescent biomolecular probes, utilizing a fluorogenic dye and biodegradable, biocompatible nanomaterials, is demonstrated. The synthesis of a new dicationic asymmetric monomethine cyanine dye with benzo[d]thiazolium-N-propionamide and chloroquinoline end groups is presented. The photophysical properties of the newly synthesized dye were examined through the combined application of spectroscopic and theoretical methods. The applicability of the dye as a fluorogenic nucleic acid probe was proven by UV-VIS spectroscopy and fluorescence titration. The dye–nucleic acid interaction mode was investigated by UV-Vis and CD spectroscopy. The newly synthesized dicationic dye, like other similar fluorogenic structures, limited permeability, which restricts its use as a probe for RNA and DNA. To enhance cellular delivery, we utilized a patented technology that employs solid, insoluble lipid nanoparticles. This method ensures the complete introduction of the dye into cells while minimizing activity outside the cells. In our study involving two human cell lines, we observed improved penetration through the cell membrane and distinctive selectivity in visualizing nucleic acids within the cytoplasm and nucleus. Full article

The self-quenching fluorogenic probe facilitates precise identification of LAMP (loop-mediated isothermal amplification) amplicons, unaffected by non-specific products resulting from primer dimers. However, low quenching efficiency by surrounding nucleobases leads to high background signal, posing significant challenges for visual inspection with the naked eye. The present study aims to identify an oligonucleotide sequence that is complementary to the self-quenching fluorogenic probe, and to employ the fluorescence super-quenching mechanism of double-stranded DNA to establish a visualization system for the LAMP assay. The results indicated that the incorporation of a sequence fully complementary to the probe could significantly reduce the system’s background fluorescence (p < 0.05). When the melting temperature exceeds room temperature, truncating the complementary sequence from the 3′ end does not compromise the probe’s quenching efficiency. The LAMP visualization system, using a 10–13-base complementary sequence of the loop primer-based probe, could effectively minimize background fluorescence and yield straightforward visual results post-reaction. Applied to rainbow trout and Atlantic salmon detection, the system detected 1 pg DNA in a closed-tube format. In conclusion, a suitable complementary sequence can reduce the background fluorescence of the self-quenching fluorogenic probe. Employing this sequence alongside the self-quenching fluorogenic probe to develop a low-background fluorescence LAMP system demonstrates great potential for successful visual detection and holds considerable promotional merit. Full article

Leaf senescence is a developmental process allowing nutrient remobilization to sink organs. Previously cysteine proteases have been found to be highly expressed during leaf senescence in different plant species. Using biochemical and immunoblotting approaches, we characterized developmental senescence of barley (Hordeum vulgare L. var. ‘GemCraft’) leaves collected from 0 to 6 weeks after the onset of flowering. A decrease in total protein and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunits occurred in parallel with an increase in proteolytic activity measured using the fluorogenic substrates Z-RR-AMC, Z-FR-AMC, and casein labeled with fluorescein isothiocyanate (casein-FITC). Aminopeptidase activity detected with R-AMC peaked at week 3 and then decreased, reaching a low level by week 6. Maximal proteolytic activity with Z-FR-AMC and Z-RR-AMC was detected from pH 4.0 to pH 5.5 and pH 6.5 to pH 7.4, respectively, while two pH optima (pH 3.6 to pH 4.5 and pH 6.5 to pH 7.4) were found for casein-FITC. Compound E-64, an irreversible cysteine protease inhibitor, and CAA0225, a selective cathepsin L inhibitor, effectively inhibited proteolytic activity with IC₅₀ values in the nanomolar range. CA-074, a selective cathepsin B inhibitor, was less potent under the same experimental conditions, with IC₅₀ in the micromolar range. Inhibition by leupeptin and phenylmethylsulfonyl fluoride (PMSF) was weak, and pepstatin A, an inhibitor of aspartic acid proteases, had no effect at the concentrations studied (up to 0.2 mM). Maximal proteolytic activity with the aminopeptidase substrate R-AMC was detected from pH 7.0 to pH 8.0. The pH profile of DCG-04 (a biotinylated activity probe derived from E-64) binding corresponded to that found with Z-FR-AMC, suggesting that the major active proteases are related to cathepsins B and L. Moreover, immunoblotting detected increased levels of barley SAG12 orthologs and aleurain, confirming a possible role of these enzymes in senescing leaves. Full article