This study presents a workflow for rapid production and functional characterization of antibody fragments using an E. coli-based cell-free protein synthesis (CFPS) system. We quantified binding interactions by fluorescence correlation spectroscopy (FCS), which enabled determination of dissociation constants (KD) between antibodies and the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. As an initial validation, two conventionally expressed anti-RBD antibodies were analyzed to establish the reliability of the FCS-based binding measurements. To enable efficient cell-free production, strategies were developed to improve the solubility and yield of single-chain variable fragments (scFvs), including implementation of an established two-stage refolding workflow adapted for CFPS-derived proteins. This approach enabled recovery of functional scFvs from insoluble fractions. In addition, Fab fragments were successfully produced and characterized, with binding measurements confirming retention of antigen recognition. Together, these results demonstrate that CFPS can be combined with FCS to enable rapid, solution-phase evaluation of antibody fragment binding without reliance on conventional cell-based expression systems. This integrated platform provides a scalable approach for antibody screening and characterization, with potential applications in therapeutic antibody development and high-throughput discovery.
Multiparametric flow cytometry (MFC) data analyses still largely rely on expert-dependent subjective manual gating strategies. While unsupervised clustering methods have improved data exploitation, no formal probabilistic decisional framework has yet been proposed for the critical subsequent step of cell subset assignment and quantitation. LBC-Flow (patent pending) is a structured naïve Gaussian Bayesian decisional framework built upon FlowSOM clustering. Unlike black-box machine learning approaches, every classification decision is fully interpretable, explicit and mathematically justified by its posterior probability value. A reference model including all cell distribution parameters must be constructed from normal body fluid, enabling the computation of posterior probabilities for each cell. Cells failing to meet predefined acceptance thresholds are flagged as a new specific class of Non-Classifiable Events (NCE). A leukocyte differential panel was used as proof-of-concept on 36 blood samples. Five analytical strategies were compared: manual gating, FlowSOM-only quantification, and three Bayesian classification approaches-(i.e. two discrete based on nodes (BDN) or cells (BDC) and one gaussian based on cells (BCGC)-. Using intraclass correlation coefficient ICC(3,1) and Z score for comparisons, BCGC demonstrated better performance than the other methods particularly for rare subsets. Complete results were delivered for 26 identified leukocyte subpopulations and NCE, in less than 30 s per sample. LBC-Flow is an original formal Bayesian decisional framework, explicitly addressing the gap between unsupervised clustering output and reproducible cell subset assignment and quantitation. Panel-agnostic by design, this approach provides a methodological foundation to be tested with other flow cytometry datasets in clinical or research contexts.
Nucleoside analogue-based RNA labelling provides powerful approaches for investigating RNA synthesis, turnover, and post-transcriptional regulation. However, conventional detection workflows often require chemical treatment, reagent removal, and repeated RNA purification, which can cause substantial sample loss and limit their application to ultralow-input samples. Here, we present a chemical conversion-coupled RT-PCR method that eliminates post-conversion RNA purification for the detection and quantification of N6-allyladenosine (a6A)-labelled RNA. Iodine-induced cyclization converts a6A into a cyclized adenosine derivative that generates characteristic cDNA mutation signatures during reverse transcription. By eliminating iodine removal, alkaline stabilization, and post-conversion RNA purification, the workflow enables reverse transcription directly from the chemical reaction mixture and reduces handling steps that may otherwise cause sample loss. Optimization of iodine treatment and reverse transcriptase compatibility identified Induro reverse transcriptase as suitable for quantitative analysis because it combined a high mutation rate with the highest estimated read-through efficiency across cyclized a6A sites. The method detected a6A-labelled RNA from femtogram-level inputs and supported mutation-rate-based quantification across a broad input range. Its applicability was further demonstrated by time-resolved tracking of IVT-generated mRNA carrying different poly(A) tails using ultralow amounts of total RNA. This workflow provides a sensitive platform for the detection and relative quantification of a6A-labelled RNA when sample availability is limited.
In the era of miniaturization, the advent of sensors and chips is rapidly increasing for systematic investigation, monitoring and diagnosing unprecedented diseases in preclinical and clinical research. Early-stage detection demands a miniaturized platform/ artificial model in clinical laboratories and healthcare centers. This enables us to investigate and study the physiological processes of the samples and the drug's efficacy on them in a cost-effective and time-efficient manner. The technology of chip systems is rapidly evolving in an attempt to bridge the gaps between preclinical and clinical studies, as well as their wide applications in research laboratories in low-resource settings. Emerging research is focusing on 3D cell culture in microfluidic devices in order to facilitate the uniform distribution of nutrients and in-vitro investigation for assessing various biological processes and phenomena due to limitations in conventional 2D cell culture. It is crucial to understand extensively the significant parameters associated with the development of microfluidic chips for culturing and forming uniform 3D cell masses. The goal of this review is to highlight the various on-chip models that offer precise control over the size of 3D aggregates and their respective microenvironment, making them suitable for drug screening and delivery in therapeutic applications. Additionally, the manuscript discusses the design considerations and feasible microfabrication techniques for generating tumor models, organoids, or organ-on-chips, and the respective computational parameters that govern nutrient flow and permeation, growth conditions, and the microenvironment, cultivating the state-of-the-art microfluidic chips for 3D cell culture.
Schiff base ligands and their coordination complexes are of significant interest due to their structural versatility and therapeutic potential. In this study, a novel Schiff base ligand, (Z)-N'-((1H-indol-3-yl)methylene)isonicotinohydrazide (HL), was synthesized with a 93% yield via the condensation of 4-pyridinecarboxylic acid hydrazide and indole-3-carboxaldehyde. Stable metal complexes of copper(II), manganese(II), and strontium(II) were subsequently prepared in a 2:1 ligand-to-metal ratio, achieving yields of 86%, 90%, and 84%, respectively. Structural elucidation was rigorously performed using elemental analysis, Fourier transform infrared (FT-IR), ultraviolet-visible spectroscopy (UV-Vis), and nuclear magnetic resonance (1H NMR) spectroscopy, confirming neutral bidentate coordination via the azomethine nitrogen and carbonyl oxygen. In addition to these methods of characterization, molecular modeling techniques, particularly density functional theory (DFT), were performed to assess the electronic structure of the novel Schiff base ligand and the metal-complexes formed with the Schiff base could be used to determine the interactions between molecular orbitals. DFT calculations (B3LYP/LANL2DZ/6-31G(d,p) with PCM solvation) revealed a reduction in the HOMO-LUMO energy gap (ΔE) from 4.16 eV in HL to 2.77 eV in HL-Sr, indicating increased chemical reactivity upon complexation and high electrophilicity, indicating good kinetic stability. In vitro antibacterial and antifungal evaluations demonstrated concentration-dependent activity; notably, metal coordination enhanced antimicrobial efficacy, with HL-Sr displaying strong antibacterial inhibition against P. aeruginosa (9.47 mm at μg/mL) comparable to the standard drug Streptomycin (10.63 mm). In vitro assays showed enhanced antibacterial and antifungal activity upon metal coordination. Although lower than reference drugs, these compounds serve as promising lead structures for future optimization rather than immediate clinical candidates. Molecular docking supported strong binding of the synthesized Schiff base metal complexes to biological targets. Molecular docking against S. aureus DNA Gyrase B and A. niger endoglucanase showed that the uncoordinated ligand HL achieved higher binding affinities (-12.11 kcal/mol and -12.60 kcal/mol, respectively) than the commercial control drugs Streptomycin (-10.90 kcal/mol) and Ketoconazole (-8.60 kcal/mol), while the metal complexes exhibited favorable interactions with key active site residues. These results highlight the synthesized compounds as promising candidates for pharmaceutical and biomedical applications.
Early diagnosis of cholangiocarcinoma (CCA) remains challenging because existing diagnostic approaches often lack sufficient sensitivity for reliable detection of early-stage disease. Circulating tumor cells (CTCs) in blood and exfoliated tumor cells (ETCs) in bile represent valuable targets for liquid biopsy-based detection; however, their low abundance and the complexity of clinical sample analysis pose substantial technical challenges for reliable enrichment and identification. Herein, we present a reproducible workflow for isolating and identifying CCA tumor cells from blood for CTCs and bile for ETCs using synthetic cell-surface heparan sulfate (HS) octasaccharide-functionalized magnetic beads (MBs) on integrated microfluidic systems. The method combined sample pre-processing, magnetic bead-based enrichment, controlled low-shear mixing and immunofluorescence-based identification into a unified workflow compatible with distinct clinical sample types. Key operational parameters, including MB concentration, mixing frequency, and pressure settings, were detailed to facilitate consistent performance. Using this workflow, tumor cell capture rates of approximately 70% in bile (for ETCs) and blood (for CTCs) were achieved, with a total processing time of 60-90 min per sample under clinically relevant low-abundance conditions. The platform enables reliable detection of as few as 1 tumor cell per mL of blood or bile. This method provides a practical and adaptable strategy for glycosaminoglycan-mediated liquid biopsy applications and may be extended to other tumor-cell enrichment workflows involving heterogeneous cell-surface interactions.
MicroRNAs (miRs) are central regulators of tumor initiation and progression, and their aberrant expression patterns have been identified as clinically valuable biomarkers for the early diagnosis of malignancies and prognostic evaluation. Here, we report tag-free fluorescence biosensing platform for the detection of circulating miRs in serum, targeting microRNA-21 (miR-21) and microRNA-10b (miR-10b) as clinically relevant oncogenic markers. The assay integrates CRISPR/Cas12a-mediated signal regulation with complementary strand (CS)-mediated target recognition. In this strategy, target miRs hybridize with the CS, thereby preventing CS-mediated activation of the Cas12a-crRNA complex. As a result, Cas12a collateral cleavage is suppressed, the G-quadruplex reporter remains intact, and Thioflavin T fluorescence is enhanced. The platform demonstrated excellent sequence discrimination capability, effectively distinguishing closely related and mismatched targets. Sensitive quantification was achieved with limits of detection of 1.4 nM for miR-21 and 852 pM for miR-10b. Importantly, robust analytical performance was maintained in complex biological matrices, confirming its applicability to serum samples. Collectively, this CRISPR/Cas12a-enabled fluorescent biosensor provides a simple approach for circulating miR detection.
DNA double-strand break (DSB) is one of the most genotoxic lesions, and unrepaired DSBs can lead to chromosomal aberrations and eventually cause carcinogenesis, ageing and dysfunctions of organs. Therefore, studies of DNA repairs and DNA damage responses (DDRs) corresponding to DSBs has been gathered intense interest. Meanwhile, understanding of the clinical symptoms how dysfunction and accelerated ageing of various organs caused by the effects of DNA damages has been less accomplished. To address this, we have developed a new method that we could investigate DSB formation in trophoblast organoids. Organoids are self-organized three-dimensional tissues that mimic the complex structures and functions of an organ but are not widely used in nucleic acids research field. Using trophoblast organoids, we have examined the DSB formation induced by the treatments with various DNA damaging agents by pulsed-field gel electrophoresis. Organoids and 2D cultured cells were treated with bleomycin, mitomycin C, camptothecin, etoposide, gemcitabine, and 5-fluorouracil, and the patterns of DSB formation in the organoids and conventional two-dimensional (2D) cultured cells were compared. The pattern of DSB formation induced by DNA damaging agents in organoids was different from that appeared in 2D cultures. To further enhance versatility, we have also established a mouse organoid system and have examined its validity. We succeeded to develop a new method that can detect damage-induced DSB formation in organoids. Application of this method would be used not only for DDRs in a tissue model but also for the risk assessment of genotoxicity.
Quantitative fluorescence imaging of formalin-fixed paraffin-embedded (FFPE) tissue is often limited by intensity heterogeneity, endogenous autofluorescence, and fixation-induced artifacts. Together, these factors reduce analytic accuracy and reproducibility. In murine skeletal muscle, dyes such as Procion Yellow (ProY) are used to identify membrane-compromised cells following injury; however, overlapping autofluorescence and uneven staining hinder reliable quantification. Existing segmentation workflows, including ImageJ-based approaches, are sensitive to these variations, and standard preprocessing methods often fail to adequately normalise fluorescence intensity across whole-slide images. Here, we present a workflow for quantitative analysis of ProY-stained FFPE skeletal muscle. The pipeline combines spectral characterisation of the dye and autofluorescence, optimised whole-slide fluorescence image acquisition, ratiometric intensity normalisation, automated segmentation using Cellpose, adaptive thresholding, and particle analysis. This approach improves segmentation robustness and consistency in highly autofluorescent FFPE tissue sections while reducing user-dependent variability. As proof-of-principle, validation in mechanically injured murine skeletal muscle demonstrated that the workflow could distinguish between different levels of tissue injury. This workflow provides a quantitative approach for fluorescence-based imaging in preclinical studies, with potential for future integration into more standardised clinical histopathology workflows. Although optimised for ProY-labelled skeletal muscle, the pipeline could be adapted to other dyes and tissue types affected by autofluorescence.
Lanthanide complexes, particularly Eu3+ β-diketonates, offer narrow-band, long-lived red emission ideally suited to sensitive fluorescence assays in complex media. The Eu(TTA)3(H2O)2 chelate combines efficient near-UV excitation, robust emission and labile coordination sites, making its signal highly responsive to molecular binding events. Sildenafil citrate is a widely used PDE-5 inhibitor whose misuse, counterfeiting and potential drug interactions demand reliable monitoring in pharmaceuticals and biological fluids. However, current chromatographic techniques are costly and labor-intensive, creating a pressing need for a simple, robust, low-cost, green, field-deployable optical method for sildenafil quantification. Near-UV excitation around 350 nm efficiently promotes ligand-to-metal energy transfer to the Eu3+ 5D0 level, producing line-narrowed 5D0→7F2 emission at ∼ 614 nm whose intensity is selectively quenched by sildenafil without disturbing its spectral fingerprint. The interaction between the Eu-TTA chemosensor and sildenafil involves a mixed mechanism of dynamic fluorescence deactivation and static "antenna" shelving, governed by photo-induced electron transfer and triplet-triplet energy transfer. Under optimized experimental conditions, the sensing protocol exhibits excellent linear Stern-Volmer behavior for sildenafil concentrations from 0.2 to 8.0 µg mL-1 (r ≈ 0.999), with a limit of detection of 0.036 µg mL-1 and a limit of quantification of 0.121 µg mL-1. Fluorimetric method coupled with SupelcleanTM LC-Florisil SPE cartridge with 2-propanol elution solvent was used for the selective determination of sildenafil in human serum and urine avoiding the presence of interference. Analysis of sildenafil in pharmaceutical formulations, spiked human urine and serum affords high overall accuracy and precision, with recoveries of 95.26-99.12 %, 97.5-102.4 % and 93.7-102.3 % and RSD values of 1.29-1.35 %, 1.16-2.01 and 1.21-2.71, respectively, while AGREE and BAGI metrics confirm the method's strong greenness and practical applicability. Consequently, the Eu-TTA-based fluorimetric probe provides a simple, sensitive, and low-cost analytical platform for sildenafil determination in pharmaceutical formulations and biological matrices, with satisfactory accuracy, precision, and practical applicability. Moreover, the system probing performance surpasses existing spectrophotometric methods and rivals chromatographic techniques in sensitivity while offering significant operational simplicity, high throughput, and cost efficiency, thus providing a green and clinically relevant alternative platform.
Hepatocellular Carcinoma (HCC) remains one of the leading causes of death across the world due to late diagnosis, unresponsiveness to treatment, and poor prognosis. The presence of a hypoxic tumor microenvironment (TME) plays an essential role in the development and progression of HCC, contributing to tumor angiogenesis, metabolic remodeling, and profound immunosuppression. Hypoxic conditions not only inhibit the activity of cytotoxic T-cells but also favor the development of tumor immunity evasion mechanisms. In light of these findings, the employment of nanomaterial-mediated oxygen delivery technologies has gained popularity due to their ability to reduce hypoxia and enhance immune response. Nanotechnologies, including oxygen-generating nanoplatforms and oxygen carriers, allow altering the composition of the TME to boost the effectiveness of treatment approaches. Current studies indicate that the combination of nanotechnology and immuno-therapeutic approaches, including immune checkpoint inhibitors, CAR-T cell therapies, and cancer vaccines, leads to a significant enhancement of the antitumor effect. These strategies have shown marked regression of the tumors and an increase in life span from the preclinical results. However, there are some issues associated with biosafety, delivery, and clinical applicability. This new strategy, where oxygen-modifying nanoparticles can be combined with immunotherapy, marks a new future for HCC management.
This study investigates the interaction between human serum albumin (HSA) and taurine (2‑aminoethanesulfonic acid) in aqueous solution using a multi-technique biophysical approach. The interaction of taurine (as an additive, supplement, or endogenous metabolite) with HSA is critically important for several interconnected scientific, pharmaceutical, and health-related reasons. This interaction was investigated using fluorescence spectroscopy, UV-visible absorption, zeta potential, surface plasmon resonance (SPR), and molecular docking under physiological conditions. Fluorescence quenching showed KSV increasing with temperature (2.99 × 103 to 5.11 × 103 M-1), indicating dynamic quenching. Binding constants (KA) increased with temperature, while KD decreased, with n ≈ 1, SPR gave a lower KA (1.7 × 105 M-1), reflecting surface sensitivity. Thermodynamic parameters (ΔH = +127.4 kJ/mol, ΔS = +0.54 kJ/(mol·K), ΔG = - 34.41 to - 40.22 kJ/mol) confirmed an endothermic, entropy-driven process governed by hydrophobic forces. Zeta potential shifted from - 39 mV to - 20 mV, and UV-visible showed hypochromism with red shift, indicating conformational changes. Docking revealed binding energy of - 4.5 kcal/mol at domain IIB (fatty acid region). Collectively, taurine binds to HSA via hydrophobic interactions with moderate, temperature-dependent affinity, offering insights into its transport and bioavailability in human circulation.
BACKGROUND:Endometrial receptivity is crucial to a successful pregnancy. Although molecular markers of endometrial receptivity can be obtained through endometrium biopsy, the prediction method is invasive and delayed. Hence, it is important and necessary to establish a less invasive and more reliable method for the determination of endometrial receptivity to increase the efficiency and effectiveness of assisted reproductive technology (ART) treatments. METHODS:This study was performed in the Centre for Assisted Reproduction of Shanghai First Maternity and Infant Hospital between June 2021 and March 2022. Samples left on the transfer catheter after cleavage stage embryos transfer (day 3, D3, presumable pre-receptivity, n = 96) and blastocysts transfer (day 5, D5, presumable receptivity, n = 127) in 223 ART patients who achieved successful clinical pregnancies subsequently were collected. The collected samples were further divided into a training set (n = 111 samples) and a test set (n = 112 samples) for endometrial receptivity prediction model training and validation, respectively. RESULTS:All the collected samples contained sufficient total RNA for further analyses. Ultimately, 57 signature genes specifically expressed in the epithelial cells and the macrophages were identified. The endometrial receptivity predictability of our model in the training set exhibited an area under the curve (AUC) of 1.00, indicating complete stratification of pre-receptive and receptive samples. Likewise, in the test set, our model achieved an AUC of 0.91 with accuracy of 0.83, specificity of 0.86 and sensitivity of 0.79. CONCLUSIONS:Our study demonstrates a non-invasive method of endometrial sample collection for accurate determination of endometrial receptivity.
Tetrodotoxin (TTX) is one of the most potent neurotoxins, with complex structures and analgesic effects. This toxin exists mainly in aquatic organisms, such as pufferfish, and has high biological activity. In this study, a novel three-tapered opto-fiber biosensor based on the localized surface plasmon resonance (LSPR) phenomenon was developed for the detection of TTX at different concentrations. A combiner manufacturing system and advanced fusion splicer processing technologies were used to produce a novel and unique tapered structure. Molybdenum disulfide (MoS2-NSs)/multi-walled carbon nanotubes (MWCNTs) were immobilized on the surface of the probe to enhance biocompatibility. This composite layer was deposited prior to gold nanoparticles (AuNPs), which not only provided sufficient space for the immobilization of nanoparticles but also increased the specific surface area for biomolecule attachment. Through the immobilization of AuNPs on the probe surface, the evanescent field can excite AuNPs to generate LSPR, thereby realizing the detection of TTX. The surface of the sensing probe was functionalized with bovine serum albumin, which significantly improved the selective recognition ability of TTX. The experimental results showed that the sensor had a sensitivity of 0.0016 nm/(ng/mL) in the linear detection range of 0-800 ng/mL, with a detection limit of 32.7 ng/mL. Additionally, the reusability, reproducibility, stability, and selectivity of the probe were tested and verified. The results for various indicators confirmed that the probe has high application potential in the field of TTX detection.
Accurate and comparable quantification of somatic mutations is essential for precision oncology, as clinical decision-making increasingly relies on the quantification of molecular biomarkers. Despite major technological advances, inter-laboratory variability and the lack of metrological traceability remain significant barriers to harmonization and confidence in mutation testing results. Reference Measurement Procedures (RMPs) represent a critical framework to address these challenges by anchoring molecular measurements to common quantitative standards. Here, we describe the development and validation of a candidate RMP for the detection and quantification of the clinically relevant NRAS p.Q61R mutation using digital PCR (dPCR). The assay was systematically optimized to maximize specificity and minimize cross-reactivity between wild-type and mutant alleles. Analytical characterization demonstrated excellent linearity across a broad range of variant allele frequencies (vAF), with a limit of detection of 0.1 %. Precision studies performed on commercially available circulating tumour DNA reference materials (RM) showed good repeatability and intermediate precision, while a full measurement uncertainty budget confirmed the robustness of the approach. Comparison with a commercial dPCR assay provided independent support for assay comparability and consistent vAF estimates across the investigated range. Preliminary inter-laboratory assessment supported transferability of the candidate RMP and comparability of the resulting measurements. Overall, this work establishes a metrologically characterized and transferable dPCR-based RMP for NRAS p.Q61R quantification. Its implementation can support the harmonization of molecular measurements, the value assignment of RM, and the alignment of routine and secondary methods, thereby strengthening the reliability of quantitative biomarker assessment in precision oncology.
Mapping protein-DNA interactions (PDIs) is essential for understanding transcriptional regulation and chromatin organization. Experimental approaches now range from in vitro assays that characterize intrinsic DNA-binding specificity to chromatin-based methods that capture protein occupancy in native genomes, as well as single-cell and single-molecule technologies that reveal regulatory heterogeneity across cells and individual chromatin fibers. These methods differ in resolution, sensitivity, input requirements, and their ability to preserve chromatin context, giving each approach distinct strengths and limitations. Here, we provide a comparative overview of major PDI technologies organized according to the biological scale at which they operate. We discuss their underlying principles, quantitative features, throughput, and key considerations for experimental design and method selection. We also review computational approaches for PDI analysis, including sequence- and chromatin-based binding prediction, multi-omics integration, and regulatory network inference. In addition, we discuss current challenges, such as platform-specific biases, sparse signals in single-cell datasets, and the lack of standardized benchmarking, and highlight future directions for improving PDI mapping and interpretation.
The integrity of human plasma and serum (P&S) is crucial for reliable biomarker discovery and validation, but it is affected by preanalytical exposure to thawed conditions (temperatures > -30 °C). The limited number of existing quality control methods for assessing plasma or serum (P/S) integrity often require resource-intensive techniques like mass spectrometry. We have developed and validated an accessible, cost-effective absorbance-based plate reader assay based on the oxidative ex vivo consumption of most small molecule thiols & disulfides (SMT&D) by albumin. The assay separates SMT&D from proteins, then uses tris(2-carboxyethyl)phosphine to reduce SMT&D, with subsequent quenching by 4-azidobenzoic acid, followed by reaction of thiols with Ellman's reagent. The method was optimized for yield, including steps to remove trace metals and minimize thiol re-oxidation. Due to inter-individual matrix effects, it was determined that measurements of each specimen before and after intentional expiration (which produce a single Δ-absorbance value, calibrated to Δ-SMT&D) are required to forensically evaluate prior exposure to thawed conditions. The assay demonstrated robust linearity, precision, and accuracy across diverse inter-individual matrices. Stability assessments at 23 °C, 4 °C and -20 °C revealed consistent exponential decay patterns in SMT&D, similar to those observed in the established LC/MS-based ΔS-Cys-Albumin assay. Estimated population reference ranges for P&S demonstrated sufficient dynamic range for the analysis of single samples with unknown histories. This potential was verified with blinded challenges. The assay provides a practical, readily accessible tool for quantifying P&S exposure to thawed conditions, facilitating improved quality control for investigators working with P&S for clinical or research purposes.
