Deciphering the complex chemical code of post-translational modifications (PTMs) is fundamental to proteomics but remains challenging due to the low abundance of modified proteins and the difficulty in resolving isobaric positional isomers. Conventional mass spectrometry and affinity-based methods often lack the sensitivity or specificity required to capture this full chemical diversity. Here, we present a general strategy for site-specific PTM recognition based on MspA nanopores engineered with polar charged residues. By systematically tuning the constriction site (position N91) with polar charged residues, we introduced specific electrostatic, electroosmotic flow and steric interactions that dramatically enhance molecular recognition capabilities. This engineered interface enabled the label-free discrimination of 10 distinct PTM types across 26 peptides, including phosphorylation, glycosylation, and-reported here for the first time via nanopore sensing-lysine crotonylation and succinylation. Furthermore, the nanopore is capable of distinguishing subtle positional isomers. Coupled with a machine learning algorithm that classifies single-molecule events with >98% accuracy, our findings establish a generalizable principle of electrostatic gating for PTM profiling, offering a versatile chemical tool for next-generation single-molecule proteomics.

Sensitive, reliable detection of molecular biomarkers in complex clinical samples through portable biosensors remains a central challenge for early cancer diagnostics. Field-effect transistor (FET) biosensors offer strong potential for miniaturized, low power sensing, yet their clinical translation is often constrained by probe instability, non-specific adsorption and Debye screening-limited signal transduction in serum. Here we present a site-specific anchored FET platform (SNAP-FET) that integrates genetic code expansion and click chemistry to immobilize nanobodies with a controlled, uniform, site-specific orientation in serum, within the Debye length for efficient signal transduction. This strategy yields attomolar-level sensitivity and stable electronic readout of endometrial cancer biomarkers directly in serum, overcoming longstanding limitations of biofluid FET sensing by coupling compact affinity probes with precision interface design. More broadly, the SNAP-FET and its portable implementation, ENDOCARE, provide a generalizable framework for next-generation biochemical sensing in point-of-care settings for early diagnostics in oncology. A platform combining site-specific probe orientation with scalable In2O3 field-effect transistors enables attomolar, serum-compatible detection of endometrial cancer biomarkers, offering a portable and highly sensitive foundation for accessible point-of-care cancer diagnostics.

Proteomics is the field of large-scale study of the structure, function, and dynamic interactions of proteins in biological systems, crucial for elucidating cellular functions and signaling networks. This field utilizes key methods such as mass spectrometry for protein identification and quantification, providing comprehensive insights into cellular function and disease mechanisms. However, traditional proteomics methods often lack the ability to monitor rapid protein activity and interactions in living systems in real time, severely limiting their application in dynamic biological processes. To overcome this limitation, fluorescent chemical probes have emerged as a powerful tool, enabling real-time visualization and monitoring of protein states in their native environments. This review comprehensively discusses the current status of fluorescent chemical probes in proteomics research, focusing on four different types of probes: Click-available probes, Photoaffinity probes, Photocatalytic probes, and Electrophile-based probes. Furthermore, it summarizes four different biological applications and highlights how each type of probe addresses specific challenges in proteomics.

Field–effect transistor (FET) biosensors have garnered significant interests in disease diagnosis. The electrostatic interaction between channel materials and probes/targets is important to the performance. However, the modulation of electrostatic interaction is difficult because it was severely attenuated in real environment, which is common in practical bioanalytical detection. With small size, aptamer possesses programmable base composition, which is unlimited by the binding sites. Herein, we systematically investigate the precise modulation of Debye length (λ D ) on the effective charge quantity of aptamer probe to regulate electrostatic interaction. We discovered that the performance achieves the optimum due to the largest quantity difference of aptamer probe effective charges when λ D approaches the minimum between aptamer probe lengths before and after binding with targets. This precise modulation is highly effective for both signal–on and signal–off detection of biomolecules such as serotonin and dopamine, exhibiting a wide linear detection range, spanning from 10 fM to 1.0 nM (10 5 ), and an ultra–low limit of detection (<10 fM). Importantly, this precise modulation exhibits practical detection improvement of dopamine secretion from living PC12 cells. Our findings provide a pioneering guideline for enhancing performance of aptamer–FET biosensors and offer in–depth understanding of probe molecular structure.

Live-cell imaging technology has revolutionized our understanding of preimplantation embryonic development, shifting the field from static morphological descriptions to dynamic functional analyses. This has tremendously advanced the fields of in vitro fertilization (IVF) and embryonic development. At the heart of this transition lies the strategic application of fluorescent probes, which provide the requisite sensitivity and specificity to resolve complex biological events. This review provides a comprehensive overview of fluorescent probe-based strategies designed to address four cardinal questions in peri-implantation embryology: genomic stability, cell fate determination, tissue morphogenesis, and embryo-maternal interactions. We systematically evaluate the chemical design principles and imaging modalities of various probes, which range from small-molecule organic fluorophores to genetically encoded reporters and nanoparticle-based sensors. Furthermore, we discuss how these tools facilitate the real-time visualization of chromosomal aberrations, lineage segregation, biomechanical forces, and enzymatic activities within the delicate embryonic microenvironment. This review summarizes methodological strategies for selecting and developing optimal probes across diverse application contexts. By identifying current technical bottlenecks and proposing future directions, such as NIR-II imaging and noninvasive labeling, it aims to drive the translation of basic embryonic research into advanced reproductive medicine.

This review examines peptide-conjugated fluorescent molecular rotors (FMRs) for protein detection. Their application are also highlighted for quantifying expression, monitoring structural alterations, and detecting intermolecular interactions.

As the fundamental executors of biological function, proteins are frequently dysregulated or differentially expressed in disease states, making them valuable biomarkers and/or therapeutic targets. Conventional approaches to monitoring the presence and activity of these proteins - including enzyme-linked immunosorbent assay, western blotting and mass spectrometry - have limited ability to provide real-time information on living cells. Fluorescence imaging overcomes these limitations by enabling selective, non-invasive and dynamic protein analysis. Molecular rotor fluorophores offer unique advantages owing to their high sensitivity to microenvironmental changes and tunable photophysical properties. Notably, these molecular rotor scaffolds can be functionalized into targeting probes that become highly emissive upon binding to specific proteins via the restriction of intramolecular rotation. Here, we introduce molecular rotor-based probes and outline their design principles and detection mechanisms. We highlight their applications in disease diagnosis and biological research, and we discuss the current challenges and prospects for clinical translation.

Long-term imaging of the protein dynamics within living cells is crucial for understanding the mechanism of disease occurrence and developing efficient diagnosis and treatment. However, the current probes suffer from a short intracellular retention and rapid clearance. Herein, we develop a covalent targeted imaging platform (CTI) for protein dynamic tracking in living cells. The peptide-conjugated probe can achieve covalent recognition of the target protein with a peptide ligand. In tandem, the maleimide group reacts with a cysteine sulfhydryl group on the target protein to form a covalent bond. This bond formation concomitantly restricts the rotation of a fluorescent molecular rotor, resulting in the activation of a long-term fluorescent signal. As a proof of concept, the probe (MXQ) successfully enabled long-term stable tracking and precise treatment monitoring of the X-linked inhibitor of apoptosis protein (XIAP) within living MCF-7 cells. We have, for the first time, reported a covalent targeted and generalizable platform for potentially accelerating its biomedical application for diverse diseases.

Abstract Telomerase is a critical enzyme that maintains chromosomal stability and cellular proliferative activity, playing a crucial role in cell division, aging, and carcinogenesis. Nucleic acid fluorescent probe technology, celebrated for its high specificity and sensitivity, has become an essential tool for telomerase detection. This review comprehensively explores the application and recent advancements of nucleic acid fluorescent probes in telomerase sensing. It focuses on the unique features and benefits of various fluorescence-based detection methods, including the design of fluorescent probes that enhance detection efficiency and accuracy. By optimizing the sequences and structures of these probes, researchers have achieved significant improvement in the sensitivity and specificity of telomerase activity detection. The comparative analysis highlights the strengths, limitations, and ideal application scenarios of different nucleic acid fluorescent probe-based methods. These methods stand out for their precision, ease of use, and adaptability, allowing researchers to choose the most appropriate approach according to their specific needs. This review provides valuable insights and perspectives for professionals in medical and chemical fields, promoting further advancements in telomerase research and related areas.

Histone methyltransferase G9a-catalyzed histone modifications serve as critical epigenetic marks that regulate diverse cellular functions. Aberrant enzymatic activities of G9a are closely associated with various human pathologies, making them promising biomarkers for disease diagnosis and therapeutic targeting. However, the lack of efficient assays for accurate and sensitive detection of G9a, especially in complex crude cellular extracts, limits elucidation of cancer-related mechanisms and advancements of therapeutic innovations. Here, a G9a detection system based on functionalized 2D solid-state nanochannel membranes through the specific recognition between small molecule Crown probes and dissociative peptide probes was developed. The Crown probe exhibited a strong binding force with the unmethylated peptide through hydrogen bonding, thereby altering the ion transport characteristics within the nanochannels and enabling the differentiation of methylated and unmethylated peptides followed by the detection of histone methyltransferase G9a. The excellent sensing performance enables diverse detection scenarios, including differentiation of different subtypes of breast cancer cells, screening of enzyme inhibitors and sensing of hypoxic cellular environments. This research proposed that the GO-Crown membrane could be an effective detection system for G9a, which provides a powerful tool for cancer research and clinical application.

Peptide self-assembly has emerged as a highly regarded strategy for developing functional agents to control cellular fate. However, due to the complexity of intracellular environments, achieving spatiotemporally traceable organelle localization and functional perturbation through peptide self-assembly remains a challenge. In this study, we developed T-FFVLK, a fluorescent peptide probe that integrates a red-emitting mitochondria-targeted molecule with a self-assembling peptide for visual tracking. This probe sequentially enters cellular compartments, ultimately targeting mitochondria to form disruptive self-assembled structures while reporting mitochondrial dysfunction. Once endocytosed into the cell, T-FFVLK initially forms short fibers that promote lysosomal escape, subsequently entering the mitochondria, where it further forms long fibers, all accompanied by a gradual increase in fluorescence and self-reporting of probe localization and mitochondrial dysfunction. T-FFVLK achieves mitochondrial enrichment within 6 h and induces morphological and functional changes within 12 h, leading to microtubule damage and cell cycle arrest. Thus, T-FFVLK is an invaluable tool for the observation of mitochondrial dysfunction in living cells, offering great potential for subcellular imaging and disease treatment.

Accurately simulating or sensitively monitoring specific substances, such as ions, molecules, and proteins in the life process, is essential for gaining a fundamental comprehension of the underlying biological mechanism, which has been a trending topic for many years. Solid-state nanochannels, inspired by biological ion channels, have been developed for decades and have achieved significant success, representing the forefront of the interdisciplinary fields of bioanalytical chemistry and nanotechnology. Typically, solid-state nanochannels with a pore size of less than 100 nm are selected to construct nanochannel-based biosensors, which can be an excellent platform to analyze small analytes, such as ions and small molecules, in a restricted space and simulate the intricate process of ion transport in living organisms. Furthermore, by integrating functional components that are termed probes into artificial devices, the nanochannel system has emerged as a remarkable tool for label-free and highly sensitive detection in practical applications. Nonetheless, the detection of large substances (more than nanoscale in size) has consistently posed a significant challenge, since previous research on solid-state nanochannels has mainly concentrated on the contribution of probes at the inner wall, which requires the biotargets to enter the nanochannel for successful detection. Moreover, the lack of testing techniques for the chemical and physical properties of probes anchored deep inside confined nanochannels results in an unclear working mechanism, which is another issue that cannot be ignored. The requirement for a more efficient and extensive detection platform has spurred an in-depth study of nanochannels, which provides innovative insight concentrating on the less restricted space on the outer surface (OS) of nanochannels and the probes at the OS (POS).In this Account, several approaches to constructing the OS and modifying POS are briefly summarized. Subsequently, ultrasensitive detection of analytes across a range of sizes, encompassing not only the ions and small molecules from ∼100 pm to ∼2 nm but also the large substances from ∼2 nm to ∼20 μm through the use of POS in the last five years, is introduced. Through the characterization of OS and the precise control of POS, the sensing mechanism, including surface charge and wettability, with POS is discussed unambiguously. Additionally, an intelligent model using dual-signal responses such as electrical and optical to enhance the responsiveness and accuracy of quantitative analysis is discussed, which can distinguish the conformation of an analyte by the exposed single cysteine thiol group. We expect that this timely Account will offer instructive insights into the development of a nanochannel-based platform to facilitate the analysis of biomolecules of varying sizes.

The trans-cleavage activity of the CRISPR-Cas system holds broad potential across diverse fields, yet precise spatiotemporal regulation remains challenging due to the predominantly single-direction control strategies available. Here, we present a bidirectional, multi-round modulation strategy for CRISPR-Cas12a trans-cleavage activity, utilizing toehold-mediated strand displacement and photocontrolled dissociation. This approach enables dynamic transitions between on and off states: Cas12a activity is activated by an activator, inhibited by a photosensitive blocker, and reactivated via UV light. We further integrated this system with DNA cryptography, establishing a hierarchical temporal authorization system that enhanced cryptographic security. The method supported multi-round modulation, achieving restoration of 95.4% activity after multiple cycles in the on state while maintaining suppression to 12.4% in the off state. This precise control strategy provides a versatile tool for spatiotemporal regulation in CRISPR-based applications, with significant implications for advanced gene editing, diagnostics, and bioengineering.

DNA molecules can be used to build "neural networks" that function like the brain, enabling them to perform complex computational tasks. However, a fundamental limitation of existing DNA networks is that their most basic computing units cannot perform true continuous and precise analog calculations, which restricts their ability to process complex information effectively. To address this, here we develop a DNA computing unit called CALCUL. This system successfully achieves fully analog computation, where all inputs, weighting parameters, and outputs are continuous and precise values. It performs the core operations of a neural network rapidly with high accuracy and is reusable. By integrating magnetic bead technology, we also enable modular operations and the construction of multilayer networks. Ultimately, we use this technology to construct a deep DNA neural network that correctly identifies complex color images with 100% accuracy. These developments provide a robust foundation for building more powerful and precise molecular computers.

Solid-state nanochannels with probe modification demonstrate effective spontaneous charge modulation and selective ionic current regulation. Outer-surface functionalization of these nanochannels enables tunable ionic current signals before and after analyte detection. To modulate local charge distributions, we designed sensing nanochannels with significant ion rectification properties for protein detection. In this work, we utilized asymmetrically charged sensing nanochannels with DNA probe modifications to generate abundant ionic current information for multianalyte recognition. During protein detection, DNA probes on the outer surfaces of the nanochannels were competitively replaced by proteins, thereby modulating the local charge distribution. This modulation influences the ionic current through ion rectification, generating cross-reactive and differentiated signals for each target. As a result, the sensing nanochannels with asymmetry of the surface charge effectively discriminate 6 proteins using only one kind of probe. Furthermore, this system successfully distinguished proteins across various concentrations and within complex environments. This work represents a significant advancement in the development of differential sensors based on outer-surface-functionalized nanochannels for multianalyte discrimination.

Hypochlorous acid exists as HClO in acidic conditions and as ClO- in alkaline conditions, posing a significant challenge for differentiation due to their strong and closely similar oxidative reaction activities. Addressing this challenge, our study presents an asymmetric donor-acceptor-donor ' (D-A-D ') molecular architecture for the design of a fluorescent probe (PMT NPs) that demonstrates exceptionally high specificity toward HClO alongside an optimized ratiometric response. The incorporation of the strong electron acceptor 2-(diphenylmethylene)malononitrile (A) modulates the reducing ability of the phenothiazine recognition site, adjusting the probe's oxidation potential to an intermediate level between HClO and ClO-. This adjustment directly dictates the probe's selectivity, enabling it to respond exclusively to HClO. By incorporating D ', the probe's response to HClO shifts the intramolecular charge transfer (ICT) from the original D-A to D '-A, instead of the usual Dox-A as presented in previous works. This adjustment controls the blue shift in fluorescence wavelength upon recognition, thereby improving the accuracy of ratiometric signals in vivo. The ability of PMT NPs to precisely recognize HClO in acidic environments was validated through live cell imaging and in vivo experiments using zebrafish and mouse models, enabling real-time monitoring of HClO surges. This dual-pronged molecular design strategy, which combines D-A interaction modulation with a D-A-D ' molecular architecture, promises to revolutionize probe designs for various biomolecules and is anticipated to advance the understanding of diseases linked to these analytes.