Search Results (123)

Background/Objectives: Rapid, equipment-free molecular detection of bacterial pathogens at the point of care (POC) remains a critical challenge. Staphylococcus aureus is a leading cause of severe infections, necessitating simple and sensitive diagnostic tools. Methods: We developed an integrated assay combining helicase-dependent amplification (HDA) and rolling circle amplification (RCA) in a sequential ‘one-pot’ format. Asymmetric HDA generates short, single-stranded amplicons from S. aureus DNA, enabling specific padlock probe ligation and subsequent exponential RCA. For equipment-free visual detection, biotin-labeled nucleotides are incorporated during RCA, and products are captured on a silica membrane and detected using a streptavidin-HRP conjugate with 3,3′,5,5′-tetramethylbenzidine substrate, producing an unambiguous blue color. Results: The assay detected as few as 10¹ genome copies of S. aureus per reaction. Evaluation against a panel of nine non-target respiratory pathogens and human genomic DNA demonstrated 100% specificity, with no cross-reactivity. The entire procedure is performed isothermally at 65 °C in a single tube with a total assay time of approximately 90 min. Conclusions: This ‘one-pot’ HDA-RCA colorimetric assay combines high sensitivity and specificity for S. aureus in a user-friendly, almost equipment-free format. Its simplicity and robust visual readout make it a promising tool for POC diagnostics in resource-limited settings, enabling rapid clinical decisions without specialized instrumentation. Full article

This paper proposes a method for E. coli detection in a microplate format using low-molecular-weight compounds that specifically interact with the lipopolysaccharides (LPSs) of E. coli cell walls. These compounds can amplify analytical signals by binding to multiple repeating cell surface structures, while the selectivity for E. coli is ensured by preliminary cultivation on selective media, such as Endo or MacConkey agar. 3-Aminophenylboronic acid (APBA) was selected as the binding reagent for detecting E. coli LPSs. Conjugates of streptavidin (STP) and bovine serum albumin (BSA) with APBA and conjugates of biotin and soybean trypsin inhibitor (STI) and BSA were synthesized. The conditions for the sequential formation of “sandwich” type complexes (BSA-APBA conjugate/E. coli/STP-APBA/STI–biotin/STP–peroxidase) and their colorimetric detection using chromogenic peroxidase substrate were determined. The detection limit was 3 × 10² cells/mL, and the range of quantitative determination covered five orders of magnitude—from 10³ to 10⁸ cells/mL. The developed assay was successfully tested using inactivated cells of pathogenic E. coli strains, confirming its potential for application. The assay was demonstrated to have universality, with the ability to detect E. coli, other bacterial pathogens, and LPS alone. This method could be adopted for the quantitative determination of different specific bacterial species using different selective media. Full article

Ursolic acid (UA), a naturally occurring pentacyclic triterpenoid, exhibits potent anticancer properties; however, its poor solubility and bioavailability limit its therapeutic application. To overcome these challenges and facilitate molecular target identification, a set of biotin-conjugated UA derivatives (5a–d) was synthesized through selective C-28 alkylation and biotinylation. The use of microwave-assisted synthesis significantly enhanced both reaction efficiency and product purity. Among the derivatives, compound 5c exhibited increased cytotoxicity and selectivity against bladder cancer cell lines, surpassing UA in its ability to induce apoptosis, generate reactive oxygen species (ROS), and halt cell cycle progression at the G1 phase. Proteomic profiling revealed that 5c interacts with proteins involved in ER stress, RNA processing, cytoskeletal remodeling, and metabolic regulation. These findings underscore the potential of biotinylated UA derivatives as multifunctional chemical probes for mechanistic studies in the development of targeted therapies for cancer. Full article

In this work, we investigated the influence of silver nanoparticle (AgNP) size (diameters of 20, 50, and 100 nm) and magnetic bead (MB) size (diameters from 100 to 4500 nm) on silver-gold galvanic exchange signal generation in magnetic electrochemical assays. Two conjugation strategies, including biotin-streptavidin interaction and a streptavidin-specific aptamer interaction, were compared to assess differences in binding chemistry and conjugation efficiency. Calibration studies showed that 50 nm diameter AgNPs provided the best sensitivity and galvanic exchange efficiency, yielding the lowest detection limits across both conjugation strategies. Larger AgNPs produced stronger signals but reached saturation rapidly, whereas smaller particles required higher concentrations to achieve equivalent silver content. Among MBs, 1000 nm beads consistently gave the highest galvanic exchange efficiency, offering sufficient surface area for AgNP loading while minimizing steric hindrance and electrode obstruction. These findings were confirmed by complementary electrochemical impedance spectroscopy, UV-Vis absorbance, and SEM imaging, which collectively demonstrated the strong influence of bead size on charge transfer resistance and conjugation efficiency. Overall, the combination of 50 nm AgNPs with 1000 nm MBs emerged as the optimal configuration, providing improved sensitivity and reproducibility. We believe these results offer valuable design guidelines for the development of next-generation aptamer-based electrochemical biosensors for biomarker detection. Full article

Objectives: Immunotherapy utilizing immune-stimulating cytokines such as IL12 holds great promise for the treatment of cancer. However, clinical use of IL12 is hampered due to severe toxicity following systemic administration. We here present a novel treatment strategy in which IL12 is chemically silenced by conjugation to PEG masks that sterically hinder the receptor binding. Subsequently, the masks can be released on demand using a bioorthogonal click reaction, cleaving the linker connecting the masks, thereby restoring the native cytokine. This “click-to-release” approach is based on the highly selective Inverse electron-demand Diels–Alder (IEDDA) pyridazine elimination reaction between a tetrazine (Tz) and a trans-cyclooctene (TCO), optimized for fast reaction kinetics and in vivo compatibility. Selective activation in the tumor microenvironment is achieved by pretargeting one component of this reaction to the tumor, triggering local activation of the masked IL12 once it is given in a secondary i.v. injection. Methods: IL12 masking and unmasking were evaluated in vitro with PAGE and HEK-Blue reporter cells and ex vivo with ELISA. Biodistribution in mice was evaluated with I-125 radiolabeling and biotin-click histochemistry. Results: Several designs were evaluated and optimized in vitro, resulting in an IL12-TCO-PEG construct that exhibited superior masking and subsequent reactivation upon reaction with a tetrazine bound to a TAG-72-targeted diabody. In tumor-bearing mice, we demonstrated that this diabody-tetrazine could efficiently pre-localize tetrazine in the tumor. Administration of IL12-TCO-PEG 24 h later afforded efficient and selective unmasking in tumors, but not in the blood. Conclusions: These results demonstrate proof of principle of the click-cleavable IL12 prodrug approach and showcase the versatility of the click-to-release reaction. Full article

We report herein the development of a polyclonal antibody against ochratoxin A (OTA) using a specially designed synthetic OTA derivative as the immunizing hapten. This OTA derivative contains a tetrapeptide linker (glycyl-glycyl-glycyl-lysine, GGGK), through which it can be linked to a carrier protein and form an immunogenic conjugate. The OTA derivative (OTA-glycyl-glycyl-glycyl-lysine, OTA-GGGK) has been synthesized on a commercially available resin via the well-established Fmoc-based solid-phase peptide synthesis (Fmoc-SPPS) strategy; overall, this approach has allowed us to avoid tedious liquid-phase synthesis protocols, which are often characterized by multiple steps, several intermediate products and low overall yield. Subsequently, OTA-GGGK was conjugated to bovine thyroglobulin through glutaraldehyde, and the conjugate was used in an immunization protocol. The antiserum obtained was evaluated with a simple-format ELISA in terms of its titer and capability of recognizing the natural free hapten; the anti-OTA antibody, as a whole IgG fragment, was successfully applied to three different immunoanalytical systems for determining OTA in various food materials and wine samples, i.e., a multi-mycotoxin microarray bio-platform, an optical immunosensor, and a biotin–streptavidin ELISA, which has proved the analytical effectiveness and versatility of the anti-OTA antibody developed. The same approach may be followed for developing antibodies against other low-molecular-weight toxins and hazardous substances. Full article

Phthalocyanines (Pcs) are well-established photosensitizers in photodynamic therapy, valued for their strong light absorption, high singlet oxygen generation, and photostability. Recent advances have focused on covalently conjugating Pcs, particularly zinc phthalocyanines (ZnPcs), with a wide range of small bioactive molecules to improve selectivity, efficacy, and multifunctionality. These conjugates combine light-activated reactive oxygen species (ROS) production with targeted delivery and controlled release, offering enhanced treatment precision and reduced off-target toxicity. Chemotherapeutic agent conjugates, including those with erlotinib, doxorubicin, tamoxifen, and camptothecin, demonstrate receptor-mediated uptake, pH-responsive release, and synergistic anticancer effects, even overcoming multidrug resistance. Beyond oncology, ZnPc conjugates with antibiotics, anti-inflammatory drugs, antiparasitics, and antidepressants extend photodynamic therapy’s scope to antimicrobial and site-specific therapies. Targeting moieties such as folic acid, biotin, arginylglycylaspartic acid (RGD) and epidermal growth factor (EGF) peptides, carbohydrates, and amino acids have been employed to exploit overexpressed receptors in tumors, enhancing cellular uptake and tumor accumulation. Fluorescent dye and porphyrinoid conjugates further enrich these systems by enabling imaging-guided therapy, efficient energy transfer, and dual-mode activation through pH or enzyme-sensitive linkers. Despite these promising strategies, key challenges remain, including aggregation-induced quenching, poor aqueous solubility, synthetic complexity, and interference with ROS generation. In this review, the examples of Pc-based conjugates were described with particular interest on the synthetic procedures and optical properties of targeted compounds. Full article

Salmonella is a rapidly spreading and widespread zoonotic infectious disease that poses a serious threat to the safety of both poultry and human lives. Therefore, the timely detection of Salmonella in foods and animals has become an urgent need for food safety. This work describes the construction of an aptamer-based sensor for Salmonella detection, using Fe₃O₄ magnetic beads and Ag@Au core–shell nanoparticles-embedded 4-mercaptobenzoic acid (4MBA). Leveraging the high affinity between biotin and streptavidin, aptamers were conjugated to Fe₃O₄ magnetic beads. These beads were then combined with Ag@4MBA@Au nanoparticles functionalized with complementary aptamers through hydrogen bonding and π-π stacking interactions, yielding a SERS-based aptamer sensor with optimized Raman signals from 4MBA. When target bacteria are present, aptamer-conjugated magnetic beads exhibit preferential binding to the bacteria, leading to a decrease in the surface-enhanced Raman scattering (SERS) signal. And it was used for the detection of five different serotypes of Salmonella, respectively, and the results showed that the aptamer sensor exhibited a good linear relationship between the concentration range of 10²–10⁸ CFU/mL and LOD is 35.51 CFU/mL. The SERS aptasensor was utilized for the detection of spiked authentic samples with recoveries between 94.0 and 100.4%, which proved the usability of the method and helped to achieve food safety detection. Full article

Background/Objectives: This study aimed to enhance the oral delivery and therapeutic synergy of atorvastatin (AT) and docetaxel (DT) through a metronomic schedule using a transporter-targeted nanoemulsion (NE), with the goal of improving antitumor efficacy and immune modulation. Methods: AT and DT were co-encapsulated in a NE system (AT/DT-NE#E) incorporating deoxycholic acid–DOTAP (D-TAP), biotin-conjugated phospholipid (Biotin-PE), and d-α-tocopherol polyethylene glycol succinate (TPGS) to exploit bile acid and multivitamin transport pathways and inhibit P-glycoprotein efflux. The optimized NE was characterized physicochemically and evaluated for permeability in artificial membranes and Caco-2/HT29-MTX-E12 monolayers. Pharmacokinetics, tumor suppression, and immune cell infiltration were assessed in vivo using rat and CT26.CL25 mouse models. Results: AT/DT-NE#E showed enhanced permeability of AT and DT by 45.7- and 43.1-fold, respectively, across intestinal cell models and improved oral bioavailability by 118% and 376% compared to free drugs. In vivo, oral metronomic AT/DT-NE#E reduced tumor volume by 65.2%, outperforming intravenous AT/DT. Combination with anti-PD1 therapy achieved a 942% increase in tumor suppression over the control, accompanied by marked increases in tumor-infiltrating CD45⁺, CD4⁺CD3⁺, and CD8⁺CD3⁺ T cells. Conclusions: Oral metronomic administration of AT/DT via a dual-transporter-targeted NE significantly improves drug absorption, tumor inhibition, and immune response. This strategy presents a safe and effective approach for colon cancer therapy, particularly when combined with immunotherapy. Full article

Oligosaccharides and glycoconjugates play essential roles in various biological processes such as cellular recognition and signaling, and thus have attracted tremendous attention in the synthetic and biological communities over the past few decades. Contributing to this field, we have achieved the synthesis of the aminoxyglycoside pentasaccharide subunit of Pseudomonas aeruginosa polysaccharide synthesis locus (Psl) exopolysaccharide through an efficient 23 step process. This pentasaccharide was designed with an aminooxy derivative at the reducing end, which was used in a 2-step oxime-based bioconjugation to the protein carrier CRM197, with an epitope ratio of 1:4. The conjugate vaccine could generate anti-Psl antibodies that could recognize P. aeruginosa PAO1 bacteria and initiate opsonophagocytic killing of the bacteria. In addition, the aminoxyglycoside could be conveniently conjugated to a bifunctional aldehyde-biotin reagent, which can be used for quantifying antibody titers in vaccination studies. Full article

Many countries have established regulatory frameworks to monitor and mitigate Salmonella contamination in poultry products. The ability to rapidly quantify Salmonella is critical for poultry processors to facilitate early detection, implement corrective measures, and enhance product safety. This study aimed to develop an Immunomagnetic Chemiluminescent Assay (IMCA) for the quantification of Salmonella Typhimurium in ground chicken. Immunomagnetic microbeads functionalized with monoclonal antibodies were employed to selectively capture and concentrate Salmonella from ground chicken samples. A biotin-labeled monoclonal antibody, followed by an avidin-horseradish peroxidase conjugate, was used to bind the captured bacteria and initiate a chemiluminescent reaction catalyzed by peroxidase. Light emission was quantified in relative light units (RLUs) using two luminometers. Ground chicken samples were inoculated with a four-strain S. Typhimurium cocktail ranging from 0 to 3.5 Log CFU/g. Bacterial concentrations were confirmed using the Most Probable Number (MPN) method. Samples underwent enrichment in Buffered Peptone Water (BPW) supplemented with BAX MP Supplement at 42 °C for 6 and 8 h before analysis via IMCA. A linear regression analysis demonstrated that the optimal quantification of Salmonella was achieved at the 8 h enrichment period (R² ≥ 0.89), as compared to the 6 h enrichment. The limit of quantification (LOQ) was determined to be below 1 CFU/g. A strong positive correlation (R² ≥ 0.88) was observed between IMCA and MPN results, indicating methodological consistency. These findings support the application of IMCA as a rapid and reliable method for the detection and quantification of Salmonella in ground chicken. Full article

The aim of this study was to investigate the usefulness of human embryonic kidney HEK293 cells as a model of normal cells in biotin-mediated therapy. The expression and role of sodium multivitamin transporter (SMVT) in the uptake and accumulation of free biotin, as well as cationic and neutral biotinylated PAMAM dendrimers of the fourth generation synthesized in our laboratory, were assessed in HEK293 cells in comparison to other immortalized (HaCaT) and cancer cells (HepG2, U-118 MG). The obtained data showed that a higher level of SMVT in HEK293 cells was not associated with a stronger uptake of biotin and biotinylated PAMAM dendrimers. Biotinylation increased the selective uptake of neutral dendrimers in an inversely proportional manner to the concentration used; however, the accumulation in HEK293 cells was lower than that in cells of other cell lines. The time-dependent biotin and biotinylated dendrimers uptake profiles differed significantly. Therefore, it should be assumed that the efficiency of biotinylated nanoparticles’ uptake depends on multiple cellular transport mechanisms. Toxicity tests showed significantly higher sensitivity to PAMAM conjugates for HEK293 cells than for HepG2 and HaCaT cells. Molecular modeling studies and the profile of biotin uptake suggest that not only SMVT but also monocarboxylate transporter 1 (MCT-1) may play an important role in the selective transport of biotin and biotinylated nanoparticles into cells. Due to the complexity of the problem, further studies are necessary. In summary, HEK293 cells can be considered a valuable model of normal cells in the study of biotin- targeted therapy using nanoparticles based on PAMAM dendrimers. Full article

Detecting multiple tumor markers is of great importance. It helps in early cancer detection, accurate diagnosis, and monitoring treatment. In this work, gold nanoparticles–toluidine blue–graphene oxide (AuNPs-TB–GO) and gold nanoparticles–carboxyl ferrocene–tungsten disulfide (AuNPs–FMC–WS₂) nanocomposites were prepared for labeling Carcinoembryonic antigen (CEA) antibody and Carbohydrate antigen 72–4 (CA72-4) antibody, respectively, and used as two kinds of probes with different electrochemical signals. With the excellent magnetic performance of biotin immune magnetic beads (IMBs), the biofunctional IMBs were firmly deposited on the magnetic glassy carbon electrode (MGCE) surface by applying a constant magnetic field, and then the CEA and CA72-4 antibody were immobilized on the IMBs by the avidin–biotin conjugation. The assay was based on the change in the detection peak current. Under the optimum experimental conditions, the linear range of detection of CEA is of the two-component immunosensor is from 0.01 to 120 ng/mL, with a low detection limit of 0.003 ng/mL, and the linear range of detection of CA72-4 is from 0.05 to 35 U/mL, with a detection limit of 0.016 U/mL. The results showed that the proposed immunosensor enabled simultaneous monitoring of CEA and CA72-4 and exhibited good reproducibility, excellent high selectivity, and sensitivity. In particular, the proposed multiplexed immunoassay approach does not require sophisticated fabrication and is well-suited for high-throughput biosensing and application to other areas. Full article

The association between microRNAs and various diseases, especially cancer, has been established in recent years, indicating that miRNAs can potentially serve as biomarkers for these diseases. Determining miRNA concentrations in biological samples is crucial for disease diagnosis. Nevertheless, the stem-loop reverse transcription quantitative PCR method, the gold standard for detecting miRNA, has great challenges in terms of high costs and enzyme limitations when applied to clinical biological samples. In this study, an isothermal signal amplification method based on a duplex-specific nuclease (DSN) enzyme-driven DNA walker and an improved catalytic hairpin assembly (CHA) was designed for miRNA detection. First, biotin–triethylene glycol-modified trigger-releasable DNA probes were conjugated to the streptavidin-coated magnetic beads for recognizing the target miRNA. The DSN enzyme specifically hydrolyzes DNA strands when the DNA probe hybridizes with the targeted miRNA. This recycling process converts the input miRNA into short trigger fragments (catalysts). Finally, three hairpins of improved CHA are driven by this catalyst, resulting in the three-armed CHA products and a fluorescence signal as the output. This dual-cycle biosensor shows a good linear relationship in the detection of miR-21 and miR-141 over the final concentration range of 250 fM to 50 nM, presenting an excellent limit of detection (2.95 amol). This system was used to detect miR-21 and miR-141 in MCF-7 and 22RV1 cells, as well as in 1% human serum. This system can be used to evaluate the expression levels of miRNAs in different biological matrices for the clinical diagnosis and prognosis of different cancers. Full article

Rheumatoid arthritis (RA) is a chronic autoimmune disorder that causes extensive damage to multiple organs and tissues and has no known cure. This study introduces a microfluidic detection platform that combines a microfluidic reaction chip with a micro-spectrometer to accurately detect the anti-cyclic citrullinated peptide antibody (anti-CCP Ab) biomarker, commonly associated with arthritis. The surface of the microfluidic reaction chip is functionalized using streptavidin to enable the subsequent immobilization of biotinylated-labeled cyclic citrullinated peptide (biotin–CCP) molecules through a streptavidin–biotin reaction. The modified chip is then exposed to anti-CCP Ab, second antibody conjugated with horseradish peroxidase (HRP) (2nd Ab-HRP), 3,3′,5,5′-tetramethylbenzidine (TMB), and a stop solution. Finally, the concentration of the anti-CCP Ab biomarker is determined by analyzing the optical density (OD) of the colorimetric reaction product at 450 nm using a micro-spectrometer. The detection platform demonstrated a strong correlation (R² = 0.9966) between OD and anti-CCP Ab concentration. This was based on seven control samples with anti-CCP Ab concentrations ranging from 0.625 to 100 ng/mL. Moreover, for 30 artificial serum samples with unknown anti-CCP Ab concentrations, the biosensor achieves a correlation coefficient of (R² = 0.9650). The proposed microfluidic detection platform offers a fast and effective method for accurately identifying and quantifying the anti-CCP Ab biomarker. Thus, it offers a valuable tool for the early diagnosis and monitoring of RA and its progression in point-of-care settings. Full article