Noninvasive fluorescence detection of tumor-associated biomarker dynamics provides immediate insights into tumor biology, which are essential for assessing the efficacy of therapeutic interventions, adapting treatment strategies, and achieving personalized diagnosis and therapy evaluation. However, due to the absence of a single biomarker that effectively reflects tumor development and progression, the currently available optical diagnostic agents that rely on "always-on" or single pathological activation frequently show nonspecific fluorescence responses and limited tumor accumulation, which inevitably compromises the accuracy and reliability of tumor imaging. Herein, based on intramolecular charge transfer (ICT) and twisted intramolecular charge-transfer (TICT) hybrid mechanisms, we report a tandem-locked probe, , for simultaneously specific imaging-guided tumor resection and ferroptosis-mediated tumor ablation evaluation under the coactivation of nitro reductase (NTR)/viscosity. The dual-stimulus-responsive design strategy ensures that exclusively activates near-infrared (NIR) fluorescence signals upon interaction with both NTR and elevated viscosity levels through triggering ICT on while inhibiting the TICT process. Meanwhile, functionalization with a tumor-targeting hydrophilic biotin-poly(ethylene glycol) moiety enhances tumor accumulation. The probe's dual-response and tumor-targeting design minimizes nonspecific tissue activation, allowing for precise tumor identification and lesion removal with a superior tumor-to-normal tissue (T/N > 6) ratio. More importantly, was capable of evaluating ferroptosis-mediated chemotherapeutics by real-time monitoring of the alternations of NTR/viscosity levels. The results reveal that the increased tumor signals of following the combination of ferroptosis and chemotherapy correlate well with the tumor growth inhibition, demonstrating the potential of to assess therapeutic efficacy.

In proteomics, postproline cleaving enzymes (PPCEs), such as prolyl endopeptidase (PEP) and neprosin, complement proteolytic tools because proline is a stop site for many proteases. But while aiming at using PEP in online proteolysis, we found that this enzyme also displayed specificity to reduced cysteine. By LC-MS/MS, we systematically analyzed PEP sources and conditions that could affect this cleavage preference. Postcysteine cleavage was blocked by cysteine modifications, including disulfide bond formation, oxidation, and alkylation. The last modification explains why this activity has remained undetected so far. In the same experimental paradigm, neprosin mimicked this cleavage specificity. Based on these findings, PPCEs cleavage preferences should be redefined from post-Pro/Ala to post-Pro/Ala/Cys. Moreover, this evidence demands reconsidering PPCEs applications, whether cleaving Cys-rich proteins or assessing Cys status in proteins, and calls for revisiting the proposed enzymatic mechanism of these proteases.

β-Galactosidase (β-gal) has emerged as a pivotal biomarker in primary ovarian cancer. Despite the existence of numerous fluorescent probes for β-gal activity detection, quinone methide-based immobilizing probes were shown to avoid rapid diffusion of the activated fluorophore and improve the resolution. However, the synthesis of these fluorophores, particularly near-infrared fluorophores, still exhibits lower efficiency. In this study, we introduce modular and rapidly assembled self-immobilizing fluorogenic probes, capitalizing on the proximity labeling properties of quinone methide (QM). Compared to conventional fluorescent probes, these new probes not only exhibit a fluorogenic response but also achieve permanent retention, demonstrating improved detection sensitivity, particularly after cell fixation and in vivo animal model studies. This straightforward synthesis approach holds promise for broader applications in detecting other analytes.

Electrofusion is an effective method for fusing two cells into a hybrid cell, and this method is widely used in immunomedicine, gene recombination, and other related fields. Although cell pairing and electrofusion techniques have been accomplished with microfluidic devices, the purification and isolation of fused cells remains limited due to expensive instruments and complex operations. In this study, through the optimization of microstructures and electrodes combined with buffer substitution, the entire cell electrofusion process, including cell capture, pairing, electrofusion, and precise separation of the targeted fused cells, is achieved on a single chip. The proposed microfluidic cell electrofusion achieves an efficiency of 80.2 ± 7.5%, and targeted cell separation could be conveniently performed through the strategic activation of individual microelectrodes via negative dielectrophoresis, which ensures accurate release of the fused cells with an efficiency of up to 91.1 ± 5.1%. Furthermore, the survival rates of the cells after electrofusion and release are as high as 94.7 ± 0.6% and 91.7 ± 1.2%, respectively. These results demonstrate that the in situ cell electrofusion and separation process did not affect the cell activity. This chip offers integrated multifunctional manipulation of cells in situ, and can be applied to multiple fields in the future, thus laying the foundation for the field of precise single-cell analysis.

Almost all proteins on the cell surface are modified by glycosylation. Cell surface glycoproteins participate in various cellular pathways, such as cell adhesion, cell-cell communication, and immune response. Due to their functional importance, glycoproteins on the cell surface often serve as potential therapeutic targets. Recent advancements in mass spectrometry (MS) have facilitated the characterization of glycoproteins that are generally localized on the cell surface, secreted to the extracellular environment, or found in intracellular organelles such as the endoplasmic reticulum, Golgi apparatus, and peroxisome. However, the selective characterization of glycoproteins on the cell surface remains challenging. In this study, we applied enzymatic treatment to live cells, followed by MS-based glycoproteomics analysis, to assess changes in protein glycosylation at different treatment time points as a method to identify cell surface glycoproteins. To demonstrate this approach, a renal cell carcinoma cell line, A498, was treated with glycosidases, sialidase and PNGase F, over two treatment time intervals, 2 and 24 h. Glycoproteins were identified as cell surface glycoproteins from A498 cells when enzyme treatment altered the glycosylation of the glycoproteins. The results revealed the effectiveness of integrating enzymatic treatment with MS-based glycoproteomics for analyzing cell surface glycoproteins. Our established method has demonstrated the potential applications for assessing accessibility of therapeutic targets on the cell surface over time and supporting the development of new targeted therapies.

Herein, a new strategy is employed to build a controllable thermal-coupled charge ionization (TCCI) device to elucidate the desorption-ionization mechanism of plasma ion sources. Efficient synergistic desorption and ionization are achieved within the TCCI device by independently controlling the desorption temperature and plasma charges. The TCCI device efficiently ionizes samples using abundant free electrons, charges, and active species from arc plasma. The coexistence of free electrons and hydroxide radicals confers redox capability to the TCCI system, implying the presence of a unified redox mechanism even when the arc plasma is transmitted through a metal conductor over a distance. In addition, molecular ions of the analytes facilitate the differentiation between primary and secondary amines during their analysis. Notably, the TCCI device enables a switch between hard and soft ionization by adjusting the thermal desorption temperature. At high temperatures (>400 °C), the TCCI device exhibits hard ionization characteristics, producing fragment ions beneficial for isomer discrimination. The TCCI mass spectrometry exhibits robust performance in terms of sensitivity and accuracy for detecting antibiotics and sterols in saline solutions, achieving linearity with correlation coefficients ≥0.99 and excellent reproducibility. The successful analysis of seven pharmaceuticals and four sterols in complex matrices using the TCCI device demonstrates its excellent salt and matrix tolerance. Overall, the TCCI device, with its independent control over thermal desorption and arc plasma, achieves efficient synergistic desorption and ionization, overcoming limitations in existing ionization technologies and contributing to the study of gas-phase ion dynamics and mechanisms.

Flap endonuclease 1 (FEN1) is a structure-specific DNA repair enzyme that has emerged as a potential target for cancer diagnosis and treatment. However, existing FEN1 assays often suffer from complicated reaction schemes and laborious procedures, and only a few methods are available for the detection and imaging of FEN1 in living cells. Especially, FEN1 is not exclusive to cancer cells, but it is also shared by normal cells. Consequently, the specific detection of FEN1 in cancer cells remains a challenge. Herein, we develop a simple and selective fluorescent biosensor for the specific imaging of FEN1 in cancer cells and tissues by engineering a FEN1 detection probe with a telomerase-responsive unit. In the presence of telomerase, it induces an extension reaction and subsequent intramolecular reconfiguration of the detection probe, generating a suitable branched DNA structure for FEN1 recognition and facilitating the cleavage of the flap by FEN1 for the recovery of fluorescence signal. Because telomerase is undetectable in normal cells but highly upregulated in cancer cells, the detection probe can only be activated in cancer cells to generate a high signal. This assay is quite simple, with the requirement of merely a single probe for dual enzyme recognition and signal output. With the integration of the single-molecule counting technology, this biosensor can achieve a detection limit of 1.2 × 10 U/μL, and it can accurately detect FEN1 in living cells and clinical tissues, providing a new avenue for FEN1-associated fundamental research and clinical diagnosis.

Spherical biosamples such as immunobeads, cells, and cell aggregates have been widely used in bioapplications. The bioactivity of individual spherical biosamples in highly sensitive assays and individual analyses must be evaluated in a high-throughput manner. Electrochemiluminescence (ECL) imaging was recently proposed for the high-throughput analysis of diffusive molecules from spherical biosamples. ECL imaging involves the placing of spherical biosamples on a flat electrode filled with a solution. The biosamples produce (or consume) biological/chemical molecules such as HO and O, which diffuse to form a concentration gradient at the electrode. The ECL signals from the molecules are then measured to obtain the concentration profile, which allows the flux to be estimated, from which their bioactivities can be successfully calculated. However, no studies on theoretical approaches for spherical biosamples on flat surfaces have been conducted using ECL imaging. Therefore, this paper presents a novel spherical diffusion theory for spherical biosamples on a flat surface, which is based on the common spherical diffusion theory and was designated as the extended spherical diffusion theory. First, the concepts behind this theory are discussed. The theory is then validated by comparison with a simulated analysis. The resulting equation successfully expresses the concentration profile for the entire area. The glucose oxidase activity in the hydrogel beads is subsequently visualized using ECL imaging, and the enzymatic product flux is calculated using the proof-of-concept theory. Finally, a time-dependent simulation is conducted to fill the gap between the theoretical and experimental data. This paper presents novel guidelines for this analysis.

Peroxynitrite (ONOO) is a critical biomarker associated with a wide array of diseases including cancer, inflammatory conditions, and neurodegenerative disorders. This study introduces an innovative chemiluminescence nanoprobe (CLNP) based on a bicyclic dioxetane structure, designed for highly sensitive and specific in vivo imaging of ONOO. Our CLNP demonstrates exceptional capabilities in generating high-contrast imaging of disease lesions, with applications verified across tumor models, acute inflammation, and acute liver injury scenarios. Key findings highlight the probe's rapid response to oxidative species, superior tissue penetration, and high signal-to-noise ratio, underscoring its potential for real-time diagnostic applications. This work represents an important advance in the field of diagnostic imaging using CL probes, offering promising avenues for the early detection and treatment of ONOO-related pathologies.

Flow injection analysis and liquid chromatography are frequently combined with electrochemiluminescence (ECL) for flow analysis. Almost all electrochemistry flow analyses employ traditional three-electrode electrochemical flow cells which have working electrode, counter electrode, and reference electrode; however, it is expensive and difficult to fabricate a traditional three-electrode electrochemical flow cell and inconvenient to renew the electrode. In this study, we have developed a single-electrode flow cell using commercially available conductive polyethylene film as the only electrode through potential differences induced by the electrode resistance for the first time. The single-electrode flow cell features a simple structure, easy renewal of the electrode, and low cost compared to the traditional three-electrode electrochemical flow cells. Taking the typical Ru(bpy)/oxalate ECL system as the analytical model, flow analysis of clinically important oxalate was achieved using single-electrode flow cell. A regression linear equation was obtained over the oxalate concentration ranges from 1 to 200 μM, with a detection limit of 0.92 μM. The single-electrode flow cell is promising for ECL flow analysis.

Lipid droplets (LDs) are highly dynamic organelles, undertaking many important functions such as maintaining lipid metabolism and cellular homeostasis. Traditional methods to analyze LD dynamics focus on morphological changes, while chemical dynamics cannot be easily probed with traditional analytical chemistry techniques. To overcome this challenge, we show here how our phase-guided Raman sampling method, where high-resolution phase microscopy images direct a Raman sampling beam, can perform label-free, multimodal characterization of LD dynamics in living cells at both the single-cell and single-LD levels with submicron accuracy and high temporal resolution. We demonstrate the study of the morphological-compositional dynamics of human hepatocellular carcinoma cells (PLC cells) under different environmental conditions and with and without fatty acid supplementation, providing insight into LD heterogeneity and heterogeneity of response. Finally, we introduce a measurement method for the dynamics of cell-average LD composition, which can quickly and accurately characterize the lipid dynamics at the single-cell level with <30 s temporal resolution. The results here show the promise of the phase-guided Raman sampling method for dynamic morpho-chemical profiling of organelle populations.

The proteome integral solubility alteration (PISA) assay is widely used for identifying drug targets, but it is labor-intensive and time-consuming and requires a substantial amount of biological sample. Aiming at enabling automation and greatly reducing the sample amount, we developed one-pot time-induced (OPTI)-PISA. Here, we demonstrate OPTI-PISA performance on identifying targets of multiple drugs in cell lysate and scaling down the sample amount to sub-microgram levels, making the PISA method suitable for NanoProteomics. OPTI-PISA can be implemented using only the standard equipment of a proteomics lab.

Hepatic ischemia-reperfusion injury (HIRI) and induced systemic inflammation is a time-dependent multistage process which poses a risk of causing direct hepatic dysfunction and multiorgan failure. Real-time in situ comprehensive visualization assessment is important and scarce for imaging-guided therapeutic interventions and timely efficacy evaluation. Here, a logically activatable nanoreporter (termed QD@IR783-TK-FITC) is developed for time-phase imaging quantification of HIRI and induced systemic inflammation. The nanoreporters could be used for in vivo ratiometric NIR-IIb fluorescence sensing of reactive oxygen species (ROS), which can depict the in situ hepatic ROS fluctuation for the early diagnosis of HIRI in the initial 3 h. Meanwhile, the ROS-specific reaction releases renal-clearable fluorophore fragments from nanoreporters for monitoring the systematic inflammation induced by HIRI via longitudinal urinalysis. In addition, a functional relationship between digitized signal outputs (NIR-IIb ratios, urinary fluorescence) with hepatic injury scores has been established, realizing precise prediction of HIRI severity and preassessment of therapeutic efficacy. Such a time-phased modular toolbox can dynamically report HIRI-induced systemic inflammation in vivo, providing an efficient approach for HIRI treatment.

Nowadays, aggregation-caused quenching (ACQ) of organic molecules in aqueous media seriously restricts their analytical and biomedical applications. In this work, hydrogen bond (H-bond) was utilized to resist the ACQ effect of 2,5,8-triamino-1,3,4,6,7,9,9b-heptaazaphenalene (Melem) as an advanced electrochemiluminescence (ECL) luminophore, whose ECL process was carefully studied in an aqueous KSO system coupled with electron paramagnetic resonance (EPR) measurements. Notably, the H-bond-induced Melem assemblies (Melem-H) showed 16.6-fold enhancement in the ECL signals as compared to the Melem aggregates (Melem-A), combined by elaborating the enhanced mechanism. On such basis, the effective ECL signal transduction was achieved through the specific recognition of the double-stranded DNA embedded in Melem-H assemblies (Me-dsDNA) with spike protein (SP) of coronavirus disease 2019 (COVID-19). For that, such an ECL biosensor showed a wider linear range (1.0-125.0 pg mL) with a lower limit of detection (LOD) down to 0.45 pg mL, which also displayed acceptable results in analysis of human nasal swab samples. Therefore, the work provides a distinctive insight on addressing the ACQ effect and broadening the application scope of the organic emitter and offers a simple platform for biomedical detection.

Robust annotation of compounds is a critical element in metabolomics. The C-detection NMR experiment incredible natural abundance double-quantum transfer experiment (INADEQUATE) stands out as a powerful tool for structural elucidation, but this valuable experiment is not often included in metabolomics studies. This is partly due to the lack of a community platform that provides structural information based on INADEQUATE. Also, it is often the case that a single study uses various NMR experiments synergistically to improve the quality of information or balance total NMR experiment time, but there is no public platform that can integrate the outputs of INADEQUATE with other NMR experiments. Here, we introduce PyINETA, a Python-based INADEQUATE network analysis. PyINETA is an open-source platform that provides structural information on molecules using INADEQUATE, conducts database searches using an INADEQUATE library, and integrates information on INADEQUATE and a complementary NMR experiment C -resolved experiment (C-JRES). C-JRES was chosen because of its ability to efficiently provide relative quantification in a study of the C-enriched samples. Those steps are carried out automatically, and PyINETA keeps track of all the pipeline parameters and outputs, ensuring the transparency of annotation in metabolomics. Our evaluation of PyINETA using a model mouse study showed that PyINETA successfully integrated INADEQUATE and C-JRES. The results showed that C-labeled amino acids that were fed to mice were transferred to different tissues and were transformed to other metabolites. The distribution of those compounds was tissue-specific, showing enrichment of specific metabolites in the liver, spleen, pancreas, muscle, or lung. PyINETA is freely available on NMRbox.

Peroxynitrite (ONOO) is a short-term reactive biological oxidant and plays an important role in cellular signal transduction and homeostatic regulation. However, excess ONOO is associated with neurodegenerative and cardiovascular diseases. Therefore, rapid, sensitive, and accurate assays for ONOO detection are essential for exploring its physiological and pathological function. In this work, a wavelength-shifted and ratiometric fluorescent sensing platform for ONOO is constructed by splitting green fluorescent carbon dots (G-CDs) and aggregating orange fluorescent carbon dots (O-CDs). The mixed G-CDs and O-CDs (M-CDs) show a fast and precise response to ONOO in the range of 0-250 μM, with a detection limit of 10 nM. In the linearity range within 3 μM ONOO, an obvious wavelength shift of G-CDs from 495 to 475 nm is observed owing to the oxidation and nitration of ONOO to the surface-state fluorescence of G-CDs, accompanied by the splitting of G-CDs. In the linearity range of 3-250 μM ONOO, the fluorescence of G-CDs remains constant, while the molecular-state fluorescence of O-CDs gradually quenches by the oxidation and nitration of ONOO through the fluorescence static process and induces their aggregation. Additionally, M-CDs show favorable intracellular imaging of endogenous and exogenous ONOO. This study not only presents a new fluorescence wavelength shift mechanism for ONOO sensing but also provides insights into CDs' fluorescence mechanism by exploring their morphology and structure via reacting with reactive oxygen species (ROS).

Chirality is a vital property across various domains, especially for biological activity. Herein, an enzyme-free sensing platform for monosaccharide enantiomer identification was developed by utilizing the Fabry-Pérot interferometer feature of TiO nanotube arrays modified with enantioselective metal-organic framework and glucose oxidase-mimicking Au NPs. In this design, optical property is monitored by reflective interferometric Fourier transform spectroscopy (RIFTS), a highly sensitive technique for detecting changes in the average refractive index within nanotubular structures. Using glucose (Glu) enantiomers as the model targets, after the recognition of L-/d-Glu on mesoporous homochiral MIL-101 (Fe), Au NPs anchored in MIL-101(Fe) catalyze the oxidation of Glu molecules to produce hydrogen peroxide (HO). Benefiting from the confinement effect of frameworks, MIL-101(Fe), as an artificial enzyme with excellent peroxidase-like activity, catalyzes the conversion of 4-chloro-1-naphthol (4-CN) into insoluble precipitates. These gathered precipitates effectively change the average refractive index of the interferometric substrate. Based on the variation of effective optical thickness (ΔOT) values, the enantioselective determination of l-Glu and d-Glu can be achieved. Moreover, the proposed RIFTS sensor also presents broad applicability for the identification of other monosaccharide enantiomers. As the enzyme-free homochiral interferometer is directly constructed on a Ti-metal sheet, the RIFTS platform offers a robust, sensitive, and low-cost device for monosaccharide enantiomer recognition.

Therapeutic drug monitoring (TDM), which involves measuring drug levels in patients' body fluids, is an important procedure in clinical practice. However, the analysis technique currently used, i.e. liquid chromatography-tandem mass spectrometry (LC-MS/MS), is laboratory-based, so does not offer the short response time that is often required by clinicians. We suggest that techniques based on Fourier transform infrared spectroscopy (FTIR) offer a promising alternative for TDM. FTIR is rapid, highly specific and can be miniaturized for near-patient applications. The challenge, however, is that FTIR for TDM is limited by the strong mid-IR absorption of endogenous serum constituents. Here, we address this issue and introduce a versatile approach for removing the background of serum lipids, proteins and small water-soluble substances. Using phenytoin, an antiepileptic drug, as an example, we show that our approach enables FTIR to precisely quantify drug molecules in human serum at clinically relevant levels (10 μg/mL), providing an efficient analysis method for TDM. Beyond mid-IR spectroscopy, our study is applicable to other drug sensing techniques that suffer from the large background of serum samples.

Cancer is the result of the interactions between tumor cells and tumor-specific immune responses. The current biomarkers detect tumor cells' properties, but accurate measurement of tumor-specific immunity is lacking. Most immunotherapies work by activating new effector tumor antigen-specific T cells (ETASTs) or reactivating pre-existing ETASTs' repertoire. The responses to immunotherapy depend on the increase of ETASTs. The amount of ETASTs, especially in blood, is critical for therapeutic efficacy. Distinguishing ETASTs from other T cells by their structural characteristics is difficult. Therefore, nanoparticles loading whole tumor antigens are utilized to activate broad clones ETASTs pre-existing in peripheral blood, followed by detecting them. Thus, the differences between ETASTs and other T cells are transformed to the differences between activated states and unactivated states. By measuring the markers of activated states and cytotoxic functions, we can distinguish ETASTs from other T cells. Nanoparticles loading mixed multiple allogeneic tumor tissue lysates or mixed multiple tumor cell lines can be utilized as universal nanoparticles to replace nanoparticles loading personalized tumor tissue. ETASTs (TATAN-activated CD8IFN-γ) in esophageal cancer patients are more than those in healthy people. Measurement of the ETASTs in the blood of esophageal cancer patients before and after ongoing therapy showed that ETATSs increased in the blood of patients who were responsive to immunotherapy but did not increase in the blood of nonresponders. These illustrated that therapeutic efficacy was positively correlated with the level of ETASTs in PBMC. Altogether, this study provides us a highly accurate and specific biomarker for predicting the therapeutic efficacy of cancer immunotherapy and potentially other therapies, such as radiotherapy.

Immunoassays have become essential tools for detecting infectious viruses. However, traditional monoclonal antibody-dependent immunoassays are costly, fragile, and unstable, especially in complex media. To overcome these challenges, we have developed cost-effective, robust, and high-affinity nanobodies as alternatives to monoclonal antibodies for rapid detection applications. We engineered dual-epitope nanobody (NB) pairs and incorporated them into a sandwich immunosensor design to detect transmitted rotaviruses in rectal swabs and wastewater samples. To further enhance sensitivity, we synthesized an advanced two-dimensional material, MXenes@CNTs@AuNPs, which offers an extensive specific surface area that supports the enrichment and immobilization of NBs. This integration with catalase-modified magnetic probes facilitates signal generation. Subsequently, our sensor achieved a detection limit of 0.0207 pg/mL for the rotavirus VP6 antigen, significantly outperforming commercial antigen kits with a sensitivity enhancement of 3.77 × 10-fold. The exceptional sensor performance extended to specificity, repeatability, stability, and accuracy across various sample types, establishing it as a promising tool for rotavirus detection. This research outlines a viable strategy for creating a robust and ultrasensitive analytical nanoprobe, thereby addressing the critical need for efficient and reliable viral detection methods in various environments.

This study primarily employed three techniques─electrospray-scanning mobility particle sizer (ES-SMPS), nanoparticle tracking analysis (NTA), and dynamic light scattering (DLS)─to assess multimodal samples. For monodisperse particles, both ES-SMPS (all sizes) and NTA (for particles larger than 40 nm) accurately determined the mean size, while DLS overestimated it. The ES-SMPS technique demonstrated precision in particle counting for multimodal samples, with a standard deviation of around 2.5-4%. Conversely, NTA's ability to count particles potentially leads to misinterpretation. The ES-SMPS approach could identify particle peaks in multimodal (bimodal, trimodal, and tetramodal) samples and show the relatively accurate position of the mode diameter. In contrast to ES-SMPS, DLS and NTA have weaknesses in characterizing multimodal samples. While NTA's performance depends on the optical properties of particles and cannot measure silica particles smaller than 30-40 nm, ES-SMPS is independent of light scattering and can handle particles as small as ∼13 nm. The ES-SMPS also excelled in separating particle peaks of the bimodal sample with a size interval gap of 10 nm, whereas NTA needs at least 20-50 nm depending on the particle type. To sum up, the ES-SMPS method performs better and provides more accurate measurements for characterizing multimodal samples compared to NTA and DLS.