Search Results (76)

Developing appropriate methods for real-time in situ investigation of how drugs influence signaling pathways and related biomolecules holds enormous potential for helping to provide an understanding of how anticancer drugs exert their effects. Herein, we report a smart nanoprobe, PDA-MB (DOX)-Pep, constructed on the basis of polydopamine nanoparticles (PDA NPs) modified with a dense shell of molecular beacon (MB) with embedded doxorubicin (DOX) and peptide, which can respond specifically to miRNA-34a and Caspase-3 targets. Intracellular experiments demonstrated that, in comparison to the control nanoprobe PDA-MB-Pep, the smart nanoprobe could selectively respond to miRNA-34a, facilitating the release of the embedded DOX. The released DOX subsequently activated the p53 pathway, which further upregulated miRNA-34a expression, leading to additional DOX release. This initiated a cyclical process involving the probe’s response to miRNA-34a, DOX release, p53 activation, and miRNA-34a upregulation, ultimately enhancing cell apoptosis and increasing Caspase-3 expression. The designed smart nanoprobe offers a visual approach to explore how anticancer drugs influence signaling pathways and related molecules at the cellular level. Full article

Background and Objectives: HER2-positive breast cancer accounts for approximately 20–30% of all breast cancer cases and is associated with aggressive tumor behavior. Trastuzumab emtansine (T-DM1), an antibody-drug conjugate targeting HER2, is a standard second-line therapy for patients with metastatic disease. However, the impact of HER2 immunohistochemistry (IHC) expression levels on T-DM1 efficacy remains unclear. Materials and Methods: This retrospective study examined 87 patients with HER2-positive metastatic breast cancer who received T-DM1 following trastuzumab-based therapy. Patients were divided into IHC 2+ and IHC 3+ groups. Progression-free survival (PFS) and overall survival (OS) were evaluated via Kaplan–Meier analysis, and group comparisons were conducted using the log-rank test. Results: The median progression-free survival (PFS) for the entire cohort was 7.3 months (95% CI: 5.277–9.323), with a numerically longer PFS in the IHC 3+ group (8.4 months, 95% CI: 5.915–10.952) compared to the IHC 2+ group (6.3 months, 95% CI: 4.178–8.422). However, this difference was insignificant (HR: 0.91, 95% CI: 0.61–1.35; p = 0.778). Similarly, the median overall survival (OS) was 23.3 months (95% CI: 18.039–28.495), with the IHC 3+ group exhibiting a slightly longer OS (24.5 months, 95% CI: 18.600–30.400) compared to the IHC 2+ group (23.2 months, 95% CI: 12.387–34.147). Again, this difference did not reach statistical significance (HR: 0.93, 95% CI: 0.63–1.42; p = 0.369). Conclusions: Although the association between HER2 IHC 3+ expression and longer PFS and OS is promising, the lack of statistical significance suggests that IHC-based HER2 stratification alone may not be sufficient to predict the response to T-DM1. The potential of conducting prospective studies with larger cohorts and comprehensive molecular profiling to refine predictive biomarkers for optimizing therapeutic outcomes in HER2-positive metastatic breast cancer is a beacon of hope and should be pursued with optimism. Full article

This study explores the recent advances of cellulose-based materials in the context of carbon capture and conversion amid the global imperative to reduce CO₂emissions. The review emphasizes the critical importance of selecting suitable materials for establishing a robust and secure carbon capture technology. From elucidating celluloses’ molecular structure and unique properties to detailing the advancements in CO₂ capture technologies, the narrative provides a comprehensive understanding of the intricate interplay between cellulose and sustainable CO₂ management. The exploration extends to the design and synthesis of cellulose-based adsorbents, challenges in implementation, showcasing emerging trends and potential breakthroughs. Emphasizing the significance of cellulose in the circular carbon economy, this review serves as a beacon for interdisciplinary collaboration, urging further research and implementation for a greener and more sustainable future. A comprehensive overview of recent developments on cellulose-based aerogels, films, composites, and solid adsorbents in the field of carbon capture. It further elucidates the research mechanisms involved in utilizing cellulose-based materials to convert CO₂ into formic acid, methanol, carbonate, and CO, offering detailed insights. The review concludes by addressing the challenges and key issues associated with cellulose-based materials in the context of carbon capture and utilization. Full article

MicroRNA-155-3p (miR-155-3p) is an important biomarker in various pathological conditions, including cancer, making the development of sensitive and specific detection methods crucial. Here, we present a molecular beacon (MB-G4) that underwent a conformational switch upon hybridization with miR-155-3p, enabling the formation of a G-quadruplex (G4) structure. This G4 was recognized by the fluorogenic ligand N-methyl mesoporphyrin IX (NMM), producing a fluorescence signal proportional to the target concentration, making it a new detection method. The conformational dynamics of MB-G4 were characterized through circular dichroism (CD) spectroscopy and native polyacrylamide gel electrophoresis (PAGE), confirming the transition from a hairpin structure to an RNA–DNA hybrid duplex that facilitated G4 formation. The optimization of the experimental conditions, including the potassium chloride (KCl) and NMM concentrations, ensured selective detection with minimal background signal. The detection limit (LOD) was determined to be 10.85 nM, using a linear fluorescence response curve, and the specificity studies demonstrated a clear distinction between miR-155-3p and miR-155-5p. Furthermore, MB-G4 was studied with total RNA extracted from the lung cancer cell line A549 to evaluate its detection in a more complex environment and was able to detect its target, validating its potential for biological sample analysis. Full article

Telomeres play a key role in maintaining chromosome stability and cellular aging. They consist of repetitive DNA sequences that protect chromosome ends and regulate cell division. Telomerase is a reverse transcriptase enzyme counteracts the natural shortening of telomeres during cell division by extending them. Its activity is pivotal in stem cells and cancer cells but absent in most normal somatic cells. Recent advances in biosensor technologies have facilitated the in situ detection of telomerase activity, which is essential for understanding its role in aging and cancer. Techniques such as fluorescence, electrochemistry, and DNA nanotechnology are now being employed to monitor telomerase activity in living cells, providing real-time insights into cellular processes. DNA-based biosensors, especially those incorporating molecular beacons, DNA walkers, and logic gates, have shown promise for enhancing sensitivity and specificity in telomerase imaging. These approaches also facilitate the simultaneous analysis of related cellular pathways, offering potential applications in early cancer detection and precision therapies. This review explores recent developments in intracellular telomerase imaging, highlighting innovative approaches such as DNA-functionalized nanoparticles and multi-channel logic systems, which offer non-invasive, real-time detection of telomerase activity in complex cellular environments. Full article

The Phi29 DNA polymerase is renowned for its processivity in synthesizing single-stranded DNA amplicons by rolling around a circularized DNA template. However, DNA synthesis rolling circle amplification (RCA) is significantly hindered by the secondary structure in the circular template. To overcome this limitation, an engineered circular template without secondary structure could be utilized to improve the sensitivity of RCA-based assays without increasing its complexity. We herein proposed an entropy-driven special RCA technology for the detection of HPV16 E7 gene at room temperature. The strategy is composed of a molecular beacon containing a loop region for nucleic acid target recognition and a stem region to initiate RCA. With the target analyte, the stem region of the molecular beacon will be exposed and then hybridized with a special circular template to initiate the DNA amplification. We tested different designs of the molecular beacon sequence and optimized the assay’s working conditions. The assay achieved a sensitivity of 1 pM in 40 min at room temperature. The sensitivity of this assay, at 1 pm, is about a hundred-fold greater than that of conventional linear RCA performed in solution. Our proposed sensor can be easily reprogrammed for detecting various nucleic acid markers by altering the molecular beacon’s loop. Its simplicity, rapid assay time, and low cost make it superior to RCA sensors that utilize similar strategies. Full article

The association between microRNA (miRNA) and various diseases has been established; miRNAs have the potential to be biomarkers for these diseases. Nevertheless, the challenge of correctly quantifying an miRNA arises from its low abundance and a high degree of family homology. Therefore, in the present study, we devised a chemiluminescence (CL) detection method for miRNAs, known as the hybridization chain reaction (HCR)-CL, utilizing the enzyme-free signal amplification technology of HCR. The proposed methodology obviates the need for temperature conversion and offers a straightforward procedure owing to the absence of enzymatic participation, and the lumino-H₂O₂-mediated CL reaction occurs at a high rate. The technique successfully detected 2.5 amol of the target analyte and 50 amol of miR-146b in a 1% concentration of human serum. In summary, the method developed in this study is characterized by its ease of operation, cost-effectiveness, remarkable analytical prowess, and ability to detect miRNA without the need for total RNA extraction from serum samples. This method is expected to be widely used for biological sample testing in clinical settings. Full article

Background: When a body is discovered at a crime or murder scene, it is crucial to examine the body and estimate its postmortem interval (PMI). Accurate estimation of PMI is vital for identifying suspects and providing clues to resolve the case. MicroRNAs (miRNAs or miRs) are small non-coding RNAs that remain relatively stable in the cell nucleus even after death-related changes occur. Objective: This study developed a molecular beacon probe for mmu-miR-133a-5p and assessed its use in mouse muscle tissue at temperatures of 4 °C and 21 °C to estimate the PMI. Methods: A total of 36 healthy adult male BALB/c mice were divided into 9 PMI time points (0, 2, 6, 8, and 10 days) with 3 mice per time point, and they were exposed to 4 °C and 21 °C. Next, the expression pattern of mmu-miR-133a in the skeletal muscle tissue over a 10-day PMI period was analyzed using the developed molecular beacon probe. Results: The molecular beacon (MB) probe was designed for optimal thermodynamic stability with a hairpin structure that opened in the presence of mmu-miR-133a-5p, thus separating the fluorophore from the quencher and resulting in a strong fluorescence signal at 495 nm. Fluorescence intensity increased with mmu-miR-133a-5p concentration from 1 ng/μL to 1000 ng/μL and exhibited a strong correlation (R² = 0.9966) and a detection limit of 1 ng/μL. Subsequently, the expression level of mmu-miR-133a-5p was observed to be stable in mouse skeletal muscle tissue at both 4 °C and 21 °C. Conclusions: This user-friendly assay can complete measurements in just 30 min after RNA extraction and is suitable for point-of-care testing, and it possesses the potential to improve existing complex and time-consuming methods for PMI estimation. Full article

Loop-mediated isothermal amplification (LAMP) is a highly effective molecular diagnostic technique, particularly advantageous for point-of-care (POC) settings. In recent years, LAMP has expanded to include various adaptations such as DARQ-LAMP, QUASR, FLOS-LAMP, displacement probes and molecular beacons. These methods enable multiplex detection of multiple targets in a single reaction, enhancing cost-effectiveness and diagnostic efficiency. Consequently, LAMP has gained significant traction in diagnosing diverse viruses, notably during the COVID-19 pandemic. However, its application for detecting Herpesviridae remains relatively unexplored. This group of viruses is of particular interest due to their latency and potential reactivation, crucial for immunocompromised patients, including organ and hematopoietic stem cell transplant recipients. This review highlights recent advancements in LAMP for virus diagnosis and explores current research trends and future prospects, emphasizing the detection challenges posed by Herpesviridae. Full article

Fluorescence-based aptasensors have been regarded as innovative analytical tools for the detection and quantification of analytes in many fields, including medicine and therapeutics. Using DNA aptamers as the biosensor recognition component, conventional molecular beacon aptasensor designs utilise target-induced structural switches of the DNA aptamers to generate a measurable fluorescent signal. However, not all DNA aptamers undergo sufficient target-specific conformational changes for significant fluorescence measurements. Here, the use of complementary ‘antisense’ strands is proposed to enable fluorescence measurement through strand displacement upon target binding. Using a published target-specific DNA aptamer against the receptor binding domain of SARS-CoV-2, we designed a streptavidin-aptamer bead complex as a fluorescence displacement assay for target detection. The developed assay demonstrates a linear range from 50 to 800 nanomolar (nM) with a limit of detection calculated at 67.5 nM and a limit of quantification calculated at 204.5 nM. This provides a ‘fit-for-purpose’ model assay for the detection and quantification of any target of interest by adapting and functionalising a suitable target-specific DNA aptamer and its complementary antisense strand. Full article

This study re-introduces a protein-free rapid test method for nucleic acids on paper based lateral flow assays utilizing special multichannel nitrocellulose membranes and DNA-Gold conjugates, achieving significantly enhanced sensitivity, easier protocols, reduced time of detection, reduced costs of production and advanced multiplexing possibilities. A protein-free nucleic acid-based lateral flow assay (NALFA) with a limit of detection of 1 pmol of DNA is shown for the first time. The total production duration of such an assay was successfully reduced from the currently known several days to just a few hours. The simplification and acceleration of the protocol make the method more accessible and practical for various applications. The developed method supports multiplexing, enabling the simultaneous detection of up to six DNA targets. This multiplexing capability is a significant improvement over traditional line tests and offers more comprehensive diagnostic potential in a single assay. The approach significantly reduces the run time compared to traditional line tests, which enhances the efficiency of diagnostic procedures. The protein-free aspect of this assay minimizes the prevalent complications of cross-reactivity in immunoassays especially in cases of multiplexing. It is also demonstrated that the NALFA developed in this study is amplification-free and hence does not rely on specialized technicians, nor does it involve labour-intensive steps like DNA extraction and PCR processes. Overall, this study presents a robust, efficient, and highly sensitive platform for DNA or RNA detection, addressing several limitations of current methods documented in the literature. The advancements in sensitivity, cost reduction, production time, and multiplexing capabilities mark a substantial improvement, holding great potential for various applications in diagnostics, forensics, and molecular biology. Full article

Lung cancer (LC) is recognized as one of the most prevalent and lethal cancers worldwide, underscoring an urgent need for innovative diagnostic and therapeutic approaches. MicroRNAs (miRNAs) have emerged as promising biomarkers for several diseases and their progression, such as LC. However, traditional methods for detecting and quantifying miRNAs, such as PCR, are time-consuming and expensive. Herein, we used a molecular beacon (MB) bead-based assay immobilized in a microfluidic device to detect miR-155-3p, which is frequently overexpressed in LC. The assay relies on the fluorescence enhancement of the MB upon binding to the target miRNA via Watson and Crick complementarity, resulting in a conformational change from a stem–loop to a linear structure, thereby bringing apart the fluorophores at each end. This assay was performed on a microfluidic platform enabling rapid and straightforward target detection. We successfully detected miR-155-3p in a saline solution, obtaining a limit of detection (LOD) of 42 nM. Furthermore, we evaluated the method’s performance in more complex biological samples, including A549 cells’ total RNA and peripheral blood mononuclear cells (PBMCs) spiked with the target miRNA. We achieved satisfactory recovery rates, especially in A549 cells’ total RNA. Full article

Strawberry anthracnose caused by Colletotrichum spp. has resulted in significant losses in strawberry production worldwide. Strawberry anthracnose occurs mainly at the seedling and early planting stages, and Colletotrichum siamense is the main pathogen in North China, where mycelia, anamorphic nuclei, and conidia produced in the soil are the main sources of infection. The detection of pathogens in soil is crucial for predicting the prevalence of anthracnose. In this study, a visualized loop-mediated isothermal amplification (LAMP) assay and a loop-mediated isothermal amplification method combined with a TaqMan probe (LAMP-TaqMan) assay were developed for the β-tubulin sequence of C. siamense. Both methods can detect Colletotrichum siamense genomic DNA at very low concentrations (10⁴ copies/g) in soil, while both the visualized LAMP and LAMP-TaqMan assays exhibited a detection limit of 50 copies/μL, surpassing the sensitivity of conventional PCR and qPCR techniques, and both methods showed high specificity for C. siamense. The two methods were compared: LAMP-TaqMan exhibited enhanced specificity due to the incorporation of fluorescent molecular beacons, while visualized LAMP solely necessitated uncomplicated incubation at a constant temperature, with the results determined by the color change; therefore, the requirements for the instrument are relatively straightforward and user-friendly. In conclusion, both assays will help monitor populations of C. siamense in China and control strawberry anthracnose in the field. Full article

Beverages are an integral component of a person’s food package. Various types of microorganisms widely contaminate beverages. This review presents current research data aimed at identifying dominant microorganisms in beverages and molecular methods for their detection. Wine, beer, dairy drinks, and fruit juices were selected as the main objects of the study. The most contaminated beverage turned out to be fruit juice. As a result of a large number of independent studies, about 23 species of microorganisms were identified in it. At the same time, they are represented not only by bacterial and fungal organisms, but also by protozoa. Milk turned out to be the least contaminated in terms of detected bacteria. The most common pollutants of these beverages were Staphylococcus aureus, Bacillus cereus, and Vibrio parahaemolyticus. It has been established that among pathogenic genera, Salmonella sp., Campylobacter sp. and Shigella sp. are often present in beverages. One of the main tools for the quality control of beverages at all stages of their production is different types of polymerase chain reaction. The sequencing method is used to screen for microorganisms in beverages. The range of variations of this technology makes it possible to identify microorganisms in alcoholic and non-alcoholic beverages. The high specificity of methods such as PCR-RFLP, Rep-PCR, qPCR, End-point PCR, qLAMP, the molecular beacon method, and RAPD enables fast and reliable quality control in beverage production. Sequencing allows researchers to evaluate the microbiological diversity of all the studied beverages, while PCR varieties have demonstrated different fields of application. For example, PCR-RFLP, RAPD-PCR, and PCR allowed the identification of microorganisms in fruit juices, qPCR, LAMP, and the molecular beacon method in wine, LAMP and multiplex PCR in milk, and End-point PCR and Rep-PCR in beer. However, it is worth noting that many methods developed for the detection of microbial contaminants in beverages were developed 10–20 years ago; modern modifications of PCR and isothermal amplification are still poorly implemented in this area. Full article

C. difficile infection (CDI) has an important impact on both human and animal health. The rapid detection and monitoring of C. difficile PCR-ribotypes (RTs) cause of CDI is critical to control and prevent this infection. This study reports the first application of the Molecular Beacon (MB)-based real-time PCR method in genotyping important C. difficile RTs of the main evolutionary clades. The cdtR gene was used as target and the cdtR sequences were analyzed after extraction from deposited genomes or were obtained after sequencing from strains of different origin. cdtR alleles were identified after sequence comparisons and MB-based real-time PCR assays were developed to discriminate them. In total, 550 cdtR sequences were compared, 38 SNPs were found, and five different cdtR alleles were identified. In total, one or two alleles were associated to the RTs grouped in the same evolutionary clade. A MB-based real-time assay was designed for each allele and for optimized testing of the C. difficile strains. The results obtained demonstrated that the MB-based real-time PCR assays developed in this study represent a powerful, original, and versatile tool to identify C. difficile types/clades and to monitor changes in the population structure of this important pathogen. Full article