Navigating ADME profiling challenges in microphysiological systems: Evaluation of a liver-chip model for clearance prediction.

Identification of chalcopyrite-binding peptides for flotation applications using phage display and deep sequencing.

Optimized fluorescent probes for heparan sulfate sensing in live cells and human blood.

Aberrant STAT3 activation and persistent expression of HPV16 E6E7 transcripts are pivotal drivers of cervical cancer (CaCx) progression. The present study was aimed to develop a Flow cytometry- based Florescence In situ hybridization (Flow-FISH) assay for simultaneous detection of STAT3 and HPV16 E6E7 transcripts at single-cell level. A set of 48 STAT3 multi locus probes and 18 HPV16 E6E7 probes were designed using Stellaris Probe Designer. Fluorescence microscopy using these probe sets generated discrete punctate signals for both individual and simultaneous hybridizations, enabling accurate transcript identification. Flow-cytometry analysis showed quantifiable STAT3 expression across CaCx cell lines, namely HeLa, SiHa and C33a. However, HPV16 E6E7 probes showed non-specific binding, which was addressed by redesigning the probes with increased stringency. The specificity of both probe sets was then evaluated through extensive sequence alignment against all known STAT3 transcript variants (n = 27) and 98 HPV16 isolates. The redesigned phase 2 HPV16 E6E7 probes were subsequently tested in cell lines, demonstrating robust detection in HPV16-positive SiHa and CaSki cells and complete absence of signal in HPV-negative controls (C33a, MSB1, SF21) or HPV18-positive HeLa cells. Dual-color flow cytometry enabled simultaneous quantification of STAT3 and HPV16 E6E7 transcripts in both cell lines and patient's exfoliated samples. Increased dual-positive fractions across LSIL, HSIL, and SCC samples were detected that corresponded with progressive viral oncogene activity and STAT3 co-activation. Overall, the optimized probe-based Flow-FISH assay provided a sensitive, specific, and high-throughput method for transcript-level diagnostics, with potential utility for stratifying cervical lesions.

Many published aptamer sequences selected and characterized by surface-based methods fail to show quantifiable binding in solution, indicating that their true equilibrium dissociation constants (Kd) are far higher than those originally reported. This discrepancy raises fundamental concerns about the reliability of quantitative binding studies that underpin the aptamer field. Surface-based assays enable high-throughput screening but are prone to non-specific binding that can be mistaken for true molecular recognition. Therefore, reliable Kd determination should be anchored in solution-based techniques that avoid such artifacts. A growing consensus also holds that Kd values should be verified by at least two orthogonal methods, since these constants are not measured directly but are inferred from quantitative observables in the absence of reference standards. Here, we compare the principal solution-based approaches for aptamer interactions with small-molecule and protein targets, focusing on quantitative accuracy, universality, material requirements, and feasibility. This analysis proposes a best-practice roadmap for method selection, aiming to reduce experimental workload while supporting defensible Kd determination through orthogonal confirmation. We hope this roadmap encourages community discussion and convergence toward shared practices for solution-based Kd determination.

DNA-Point Accumulation for Imaging in Nanoscale Topography (DNA-PAINT) is a versatile super-resolution technique that relies on the predictable binding kinetics between fluorescent imager strands and docking strands attached to target proteins. This makes DNA-PAINT particularly suitable for multiplexing and quantitative applications, but its performance is often limited by spurious signals from non-specific binding of imager strands. Here we describe a method to remove these non-specific binding events using a statistical test to distinguish between DNA-specific and non-specific interactions. To demonstrate the method, we imaged mosaic epithelial tissues in Drosophila melanogaster egg chambers and showed that >90% of non-specific and otherwise indistinguishable signal in the super-resolved images can be removed. This denoising improves the quality of DNA-PAINT super-resolved images and is essential for accurate measurements of spatial relationships and protein quantification.

The αvβ6 integrin has emerged as a valuable target for theranostic applications in nuclear medicine with high applicability across a variety of cancers, including head-and-neck, lung, breast, and pancreatic carcinomas. [⁶⁸Ga]Ga-Trivehexin is a prominent example of a diagnostic tracer targeting this integrin. In this work, we aimed to expand on this concept by developing FSC(PEG4-αvβ6)₃, a novel tracer that retains the Trivehexin design, but features PEGylated spacers and replaces the TRAP chelator with Fusarinine C (FSC), enabling labelling with Zirconium-89 in addition to Gallium-68. Preclinical characterization of [⁶⁸Ga]Ga/[⁸⁹Zr]Zr-FSC(PEG4-αvβ6)₃ included affinity determination towards the αvβ6 integrin and cellular uptake studies in αvβ6-positive H2009 cells. A subcutaneously xenografted H2009 tumor model was used to assess the PET imaging potential and biodistribution at early time points with the Gallium-68 labelled compound, and at later time points (up to 6 days post-injection) with the Zirconium-89 labelled version. While [⁶⁸Ga]Ga-FSC(PEG4-αvβ6)₃ exhibited moderate binding to αvβ6, its affinity, cellular internalization, and tumor uptake in vivo were lower compared to [⁶⁸Ga]Ga-Trivehexin. Notably, this decreased target engagement was associated with reduced nonspecific binding, which we primarily attributed to the incorporation of PEGylated linkers. Despite indication of in vivo degradation of [⁸⁹Zr]Zr-FSC(PEG4-αvβ6)₃, still a meaningful evaluation of pharmacokinetics and biodistribution at extended time points was feasible, revealing prolonged tumor persistence up to 6 days post-injection. FSC(PEG4-αvβ6)₃ represents the first-generation tracer targeting the αvβ6 integrin based on the multifunctional chelator Fusarinine C, thereby expanding the repertoire of radionuclides applicable from Gallium-68 to Zirconium-89. Following further optimization, this novel class of compounds holds significant promise for enabling clinical translation and advancing the development of next-generation αvβ6-directed imaging agents.

The liquid–liquid phase-separated environment of the eukaryotic nucleolus is proposed to benefit ribosome assembly by chaperoning rRNA folding and spatially sorting ribosome assembly factors. Yet, how microscopic interactions within the condensed nucleolus affect rRNA folding and assembly is largely unknown. We used single-molecule fluorescence microscopy to monitor folding of the Tetrahymena ribozyme and an rRNA domain inside and outside droplets of Nop1/fibrillarin, a major constituent of the nucleolus. We found that Nop1 destabilizes tertiary docking of the ribozyme substrate helix equally in dilute and condensed phases, depending only on transient molecular interactions between Nop1 and the ribozyme. Nop1 binding also inhibits rRNA unwinding by a DEAD-box helicase and nonspecific binding of a ribosomal protein. Over time, nonspecific interactions with Nop1 are outcompeted by specific RNA-protein assembly. Our results illustrate how RNA-binding proteins residing in the nucleolus tune RNA folding stability while permitting assembly of native ribosomal complexes.

Exploring biosensors: Distinctive features and emerging applications.

Structure-affinity balance of anti-cardiac troponin I aptamer: Effects of sequence truncation.

Protein-capped mesoporous silica SBA-15 enables protease-responsive and controlled antimicrobial peptide delivery.

Orientational probes-powered sensitive and accurate immunochromatographic assays with reduced antibody consumption and diminished matrix interference.

A modular platform for surface-bound biosensing: SpyCatcher-Mediated functionalization of antifouling polypeptide brushes on gold and polystyrene.

An electrocatalytic dual molecularly imprinted polymer-based biosensor enables femtomolar detection of CD44 without antibodies.

Application of modelling of cell monolayer permeation data to generate input parameters compatible with in vitro-in vivo translation of blood-brain-barrier disposition of drugs.

Eco-friendly high-performance aptasensor for ultra-sensitive methamphetamine detection in biofluids using carbon felt modified by Ti3C2Tx MXene and silver nanodendrites.

Biomimetic membrane interface engineering constructs functionalized detection platforms by incorporating natural cell membranes, synthetic lipids, or hybrid membranes. The primary purpose of this strategy is to minimize background interference while leveraging intrinsic membrane properties for effective target interaction. Applications are diverse, ranging from the high-purity separation of circulating tumor cells (CTCs) to the efficient isolation of extracellular vesicles (EVs), as well as the subsequent detection of EV-derived contents (e.g., miRNA, protein, and mRNA) via membrane fusion mechanisms. Conceptually, this approach serves as a robust bridge between synthetic materials and biological systems. Its major advantages lie in the significant reduction of non-specific binding and the unique capability to facilitate both target capture and internal cargo analysis. However, challenges such as complex preparation processes, stability issues, and the dependence on functional modifications to address significant tumor heterogeneity remain to be resolved. This review summarizes recent progress, analyzes these critical issues, and outlines future directions.

Despite substantial progress in biosensor development, achieving reliable sensitivity and selectivity under real-world conditions remains challenging, particularly in complex and heterogeneous sample matrices. While sensitivity has historically been the primary focus of biosensor optimization, selectivity often emerges as a limiting factor for practical performance when deployed outside controlled laboratory environments. Poor selectivity can lead to false-positive or false-negative results, thereby undermining the reliability and accuracy of biosensing platforms. A major contributor to this problem is nonspecific binding, the unintended interaction between biosensor and nontarget species. However, the origins, mechanism, and implications of nonspecific binding remain insufficiently understood and are still actively debated within the scientific community. In this review, we trace the conceptual development of nonspecific binding and critically examine its physicochemical origins in substrates and biorecognition elements. We then assess recent progress in recognition elements, such as antibodies, aptamers, and enzymes, emphasizing not only their strengths but also their limitations and vulnerability to off-target interactions. To mitigate nonspecific binding, we summarize a range of emerging strategies, including optimizing the conjugation and orientation and increasing binding site accessibility and density through structural design, removing interfering species, and implementing signal-level strategies. Finally, we outline persisting challenges and future directions for enhancing biosensor selectivity. Collectively, these insights offer a roadmap for designing next-generation biosensors with high accuracy, robust selectivity, and real-world applicability.

Antisense oligonucleotides (ASOs) are an important therapeutic modality for neurological diseases. Some ASOs cause transient neurobehavioral responses acutely following intrathecal delivery to the central nervous system (CNS). We characterized a subset of these responses, including hypoactivity, spinal reflex loss, paresis, sedation, and ataxia, that are suggestive of neuronal inhibition in rodents and non-human primates. Across species, inhibition-like responses peaked ∼3 h post-ASO delivery, reversed within 24 h with no sequelae, and could be quantified using simple neurobehavioral scales. Acute inhibition was dose-responsive and was abrogated with lower phosphorothioate and guanine content in ASOs. Acutely inhibitory ASOs transiently disrupted motor pathway neurotransmissionin vivo and suppressed firing in primary neural cultures. In vitro firing rate suppression of >60% predicted high in vivo acute inhibition scores and was reversed immediately with addition of varying excitatory agents or upon ASO washout. Peak acute inhibition in vivo coincided with peak CNS tissue concentrations of ASO and abated as ASO was internalized by parenchymal cells and cleared from the extracellular space. We propose transient high extracellular concentrations block synaptic transmission via non-specific protein binding of phosphorothioate ASOs. Our results define a comprehensive framework for quantifying and mitigating ASO-mediated acute inhibition.

This paper presents the optimization of surface modification for aptameric graphene nanosensors for the measurement of biomarkers in undiluted physiological media. In these sensors, graphene transduces the binding between an aptamer and the intended target biomarker into a measurable signal while being coated with a polyethylene glycol (PEG) nanolayer to minimize nonspecific adsorption of matrix molecules. We perform a systematic study of the aptamer and PEG attachment schemes and parameters, including the impact of the serial or parallel PEG–aptamer attachment scheme, PEG molecular weight and surface density, and aptamer surface density on the sensor behavior, such as the responsivity to biomarker concentration changes, and importantly, they are used for operation in physiological media and have the ability to reject nonspecific binding to interfering molecules. We then use the understanding from this parametric study to identify graphene nanosensor designs that are optimally functionalized with PEG and aptamers to be strongly responsive to target biomarkers and effectively reduce nonspecific adsorption of interferents, thereby enabling sensitive and specific biomarker measurements in undiluted physiological media. The experimental results show that nanosensors that were optimized via serial modification with 5000 Da PEG at 15 mM and a 94 nt DNA aptamer at 500 nM allowed specific measurement of C-reactive protein (CRP) in undiluted human serum with a limit of detection (LOD) down to 27 pM, representing an up to 1000-fold improvement compared to previously reported CRP measurements.