Surface plasmon resonance (SPR) is a sensitive, label-free method for measuring biomolecular interactions, widely used in drug discovery, including fishing active components from traditional Chinese medicine (TCM). However, classical SPR systems encounter limitations when dealing with the complexity of TCM, specifically regarding high-throughput processing and distinguishing substances with different binding kinetics. To overcome these challenges, we constructed an upgraded fluidic system (UFS) on the SPR platform, integrating a customized ligand fishing sensor chip, an optimized sample flow path, and a selectable recovery mode. By screening for tumor necrosis factor receptor 1 (TNFR1) ligands, we identified esculetin as a direct binding component. In SH-SY5Y cells, esculetin targets TNFR1, reducing endogenous ligand binding and decreasing caspase-3 activity, thereby inhibiting apoptosis. The UFS system significantly enhances screening efficiency for complex natural compounds. This study provides the first evidence that esculetin directly interacts with TNFR1 via a novel anti-apoptotic mechanism, complementing its known anti-inflammatory effects and offering a valuable tool for TCM research.

Hepatocellular carcinoma, the third leading cause of cancer-related deaths globally, presents a critical public health burden in China due to its high incidence and mortality. While targeted therapies and immunotherapies have improved survival in advanced HCC, drug resistance remains a major therapeutic challenge. Recent studies suggest that gefitinib, an EGFR inhibitor, overcomes lenvatinib resistance, yet its mechanistic underpinnings are incompletely understood. To investigate gefitinib's metabolic effects in HCC, we conducted untargeted metabolomic profiling using two separate platforms: gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) with both hydrophilic interaction liquid chromatography (HILIC) and reversed-phase modes. Raw data were processed by Mass Hunter, normalized with internal standards, and analyzed via SIMCA for pattern recognition. Principal component analysis (PCA) of quality control samples and experimental groups (n = 6 each) confirmed system stability and clear inter-group separation. Orthogonal projections to latent structures discriminant analysis models were validated by 200 permutation tests. Analysis identified 42 metabolites with VIP > 1, of which 25 showed significant alterations (p < 0.05) post-gefitinib treatment. KEGG/RaMP-DB enrichment revealed perturbations in four key pathways: arginine-proline metabolism, nitrogen metabolism, branched-chain amino acid biosynthesis, and taurine metabolism. These results delineate gefitinib-induced metabolic reprogramming in HCC cells, providing a foundation for targeting metabolic vulnerabilities to overcome therapy resistance.

This study aims to develop an efficient and accurate method for detecting human serum albumin (HSA) in urine using lateral flow immunochromatography analysis (LFIA) and a smartphone-based application (App). First, a LFIA test strip using colloidal gold as the labeling material was developed and optimized. A detecting accessory was designed and produced, including a strip kit that can package the LFIA test strip, and a test cartridge that can mount the smartphone on top of it. Then an App was constructed to automatically identify the concentration of HSA on the test strip using photos taken by smartphone. The detection limit of HSA by the proposed platform is 2 µg/mL, and the quantitative range is 5 200 µg/mL. The coefficient of variation is less than 18.3

Surface plasmon resonance (SPR) is a sensitive, label-free method for measuring biomolecular interactions, widely used in drug discovery, including fishing active components from traditional Chinese medicine (TCM). However, classical SPR systems encounter limitations when dealing with the complexity of TCM, specifically regarding high-throughput processing and distinguishing substances with different binding kinetics. To overcome these challenges, we constructed an upgraded fluidic system (UFS) on the SPR platform, integrating a customized ligand fishing sensor chip, an optimized sample flow path, and a selectable recovery mode. By screening for tumor necrosis factor receptor 1 (TNFR1) ligands, we identified esculetin as a direct binding component. In SH-SY5Y cells, esculetin targets TNFR1, reducing endogenous ligand binding and decreasing caspase-3 activity, thereby inhibiting apoptosis. The UFS system significantly enhances screening efficiency for complex natural compounds. This study provides the first evidence that esculetin directly interacts with TNFR1 via a novel anti-apoptotic mechanism, complementing its known anti-inflammatory effects and offering a valuable tool for TCM research.

Surface plasmon resonance (SPR) biosensor has emerged as a transformative tool in high-throughput drug screening and label-free analysis of biomolecular interactions. However, a critical limitation of SPR lies in its stringent requirement for highly purified proteins to ensure reliable quantification of binding affinities and kinetic parameters. In order to address the limitations, the lentiviral particle and styrene-maleic acid polymer have been previously developed to extract and stabilize transmembrane proteins (TMs) indirectly and thus to detect ligands interaction with TMs by SPR biosensor. The present study proposes a high-throughput SPR-based drug screening system that utilizes cell-free protein synthesis (CFPS) to achieve in situ purification and immobilization of TMs on SPR biosensors. First, C−X−C chemokine receptor 4 (CXCR4) protein with His-tag was prepared by CFPS. Then, two types of nickel-nitrilotriacetic acid (Ni-NTA) biosensors, modified with carboxymethylated dextran (CMD) and coated with polycarboxylate hydrogel coating (HC) matrix, were compared with classical carboxymethylated dextran 5 (CM5) biosensor in order to determine the optimal strategy for coupling the CXCR4-CFPS protein. The CMD-NTA/SPR biosensor was next applied to screen for CXCR4 ligands from 96 natural products. Finally, glycyrrhizic acid and ginsenoside Re were proved to be function of CXCR4 inhibitors by affinity test, molecular docking, and cell migration assay. The combination of CFPS with SPR technology facilitates in-situ purification and immobilization of target proteins in a single step, thereby significantly enhancing the efficiency of SPR assay procedures. The system has broad applicability for targeting various challenging TMs and provides potential candidates for subsequent drug development.

The application of Bio-Layer Interferometry (BLI) is contingent upon the immobilization of highly purified target proteins onto the sensor. The cumbersome and time-consuming nature of traditional protein expression and purification processes restricts the application of BLI in high-throughput screening of traditional Chinese medicine (TCM). This study aims to develop a rapid and efficient BLI-based platform for screening bioactive components in TCM. An integrated platform combining cell-free protein synthesis (CFPS), BLI, and ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF/MS) was established for efficient TCM bioactive compound discovery. Functional C-X-C chemokine receptor 4 (CXCR4) was synthesized in vitro using a CFPS system, which were then validated by surface plasmon resonance (SPR) and western blotting. Immobilized CXCR4 on NTA biosensors enabled BLI-based high-throughput screening of TCM extracts, followed by target-specific compound recovery and characterized via mass spectrometry. Three bioactive TCM constituents were successfully fished and identified as coptisine, ligustilide, and senkyunolide A. All of them exhibited negligible cytotoxicity at concentrations ranging from 6.25 to 100 μM). Furthermore, ligustilide and senkyunolide A demonstrated certain affinity for CXCR4 with KD of 69.86 μM and 14.7 μM, respectively, and significantly inhibited cell migration. This study is the first identification of ligustilide and senkyunolide A as functional ligands of CXCR4. The established CFPS-BLI-UHPLC-QTOF/MS platform enables efficient discovery of low-toxicity, high-affinity CXCR4-targeting therapeutics from TCM.

Cholangiocarcinoma (CCA) is an aggressive malignancy with a poor prognosis. Nitidine chloride (NitC), a bioactive alkaloid derived from Zanthoxylum nitidum (Roxb.) DC., exhibits potential anti-cancer activity against CCA. However, the metabolic mechanism underlying the anti-cancer effect of NitC remains poorly understood and requires further elucidation. This study integrated metabolomics and network pharmacology to systematically investigate the anti-cancer activity and underlying mechanisms of NitC in treating human cholangiocarcinoma cells. First, The effects of NitC on human cholangiocarcinoma cells were assessed by cell proliferation, apoptosis, and cycle. Then, potential mechanisms and targets were investigated using a combination of cell metabolomics and network pharmacology and verified by molecular docking. Finally, we measured the protein levels of potential targets in TFK1 cells using enzyme-linked immunosorbent assay (ELISA). Our results indicated that NitC treatment induced the proliferation inhibition, G2/M arrest and apoptosis of TFK1 cells in a concentration dependent manner. The metabolomics analysis identified forty differential metabolic biomarkers and five key metabolic pathways of NitC in treating CCA. Network pharmacology found 36 potential targets for NitC intervention on CCA. The integration of network pharmacology and metabolomics constructed the "compound-reaction-enzyme-gene" association and revealed that NitC exerts its efficacy on CCA through four key targets, eleven metabolic indicators, and glycine, serine and threonine metabolism, and tyrosine metabolism. Molecular docking further confirmed robust binding interactions between NitC and these key targets. Moreover, ELISA results showed that NitC treatment significantly attenuated the protein levels of PIK3CA, PTGS2, and PRKACA in TFK1 cells. This study demonstrates that combining metabolomics and network pharmacology provides a powerful strategy to elucidate the pharmacological mechanisms of natural compounds, also offering new insights into the therapeutic potential of NitC for CCA.

Sini Decoction (SNT) is a traditional formula recognized for its efficacy in warming the spleen and stomach and dispersing cold. However, elucidating the mechanism of action of SNT remains challenging due to its complex multiple components. This study utilized a synergistic approach combining two-dimensional fluorescence difference in gel electrophoresis (2D-DIGE)-based drug affinity responsive target stability (DARTS) with label-free quantitative proteomics techniques to identify the direct and indirect protein targets of SNT in myocardial infarction. The analysis identified 590 proteins, with 30 proteins showing significant upregulation and 51 proteins showing downregulation when comparing the SNT group with the model group. Through the integration of 2D-DIGE DARTS with proteomics data and pharmacological assessments, the findings indicate that protein disulfide-isomerase A3 (PDIA3) may serve as a potential protein target through which SNT provides protective effects on myocardial cells during myocardial infarction.

Surface plasmon resonance (SPR) biosensors have been applied in various fields with the advantages of being label-free, having high specificity, having high sensitivity, and providing real-time monitoring. With the gradual improvement of SPR technology, SPR biosensors have been used for the detection of macromolecules such as proteins, peptides, and nucleic acids. Antibodies are generally used as the recognition component of SPR biosensors due to the high specificity of antibody–antigen binding. Recently, aptamers have become new choices instead of antibodies for their characteristic of high specificity with target molecules, high stability of chemical synthesis, convenience in storage, and ease of labeling. In this study, an aptamer-based SPR biosensor for chloramphenicol (CAP) detection was established through optimizing the conditions of CAP aptamer immobilization and analysis procedure, including biosensor type, signal enhancement, running buffer, sample diluent, and dissociation time. The results suggested that the optimal immobilization strategy of aptamers on the SPR biosensor was indirect immobilization based on the CM5 chip. The aptamer-based SPR biosensor had good specificity for CAP and could be used to detect CAP in real samples such as milk. Therefore, SPR biosensors have great application prospects in the food safety field, and aptamers deserve further study to improve the performance of the biosensor.

Acetylcholinesterase (AChE) plays a crucial role in the activities of the nervous system, and its abnormal function can lead to the occurrence and development of neurodegenerative diseases. Hence, an effective method for real-time monitoring of AChE activity is essential. Very recently, several fluorescence sensors have been developed for the detection of AChE activity, but they are usually imaging in the visible region, relatively small Stokes shifts, or long response times, limiting their application for real-time monitoring in vivo. Inspired by that, a near-infrared (NIR) off-on probe ((E)-4-(2-(4-(dicyanomethylene)-4H-chromen-2-yl)vinyl)phenyl dimethylcarbamate, DCM-N) for AChE monitoring with high selectivity and sensitivity is developed. In the probe DCM-N, a bright near-infrared fluorescence emission at 700 nm can be triggered by AChE through the cleavage of amino ester bond in DCM-N, and the resulting fluorescence exhibits a good linear relationship with AChE activity in the range of 0.2-16 U/mL, with a detection limit as low as 0.06 U/mL. For real plasma sample detection, DCM-N demonstrates advantages of accurate detection and fast response compared to the traditional Ellman assay for AChE detection. Moreover, DCM-N can be used for imaging of AChE activity in live cells and tracking of AChE activity in zebrafish models, which is of great significance for medical and physiological research related to AChE. DCM-N possesses several notable features such as light-up NIR emission, fast response, large spectral shifts and strong photostability under physiological conditions. These features enable it to monitor AChE activity both in vivo and in vitro, providing a suitable tool for real-time monitoring and in vivo visualization of AChE activity.

The study aims to promote a network toxicology strategy to efficiently investigate the underlying neurotoxicity molecular mechanisms of domoic acid（DA）, which is one of the main toxins of paralytic amnesic shellfish poisoning and has gained significant attention due to its ability to induce neurotoxicity. By utilizing ChEMBL, CTD, Drug bank, TTD, DGIdb, Pharmapper and GeneCards databases, we identified 73 potential targets associated with DA-exposure related amnesia and neurotoxicity. Further refinements via STRING and Cytoscape software highlight the protein-protein interactions. 30 targets were recognized by both the K-means algorithm and topological analysis. GO and KEGG pathway analysis conducted through DAVID databases reveals that these targets of amnesia and neurotoxicity are predominantly enriched in multiple pathways. AKT1 was identified by a multiple-topically methods as the key target. Molecular docking and bio-layer interferometry were conducted to confirm the binding between these targets and DA(-CDOCKER_INTERACTION_ENERGY =45.719 kcal/mol, KD=2.0E-11M). This research provides a theoretical basis for understanding the molecular mechanism of DA-induced neurotoxicity, as well as establishing a foundation for the prevention and treatment of DA exposure.

Bevacizumab, a monoclonal antibody targeting vascular endothelial growth factor A (VEGFA), is a widely used anti-angiogenic drug for malignancies. Its complex pharmacokinetics causes significant inter-individual concentration variations, making therapeutic drug monitoring (TDM) essential. Conventional liquid chromatography-tandem mass spectrometry (LC-MS/MS) and enzyme-linked immunosorbent assay (ELISA) for the TDM of bevacizumab suffer from limitations such as complex sample preparation and large sample volume requirements, highlighting the need for novel detection technologies. In this study, a surface plasmon resonance (SPR) biosensor for quantifying bevacizumab in human serum was developed. VEGFA was immobilized on a CM5 sensor chip, demonstrating high activity and specificity for bevacizumab. To mitigate nonspecific interference from the serum matrix, bovine serum albumin (BSA) was immobilized on the reference flow cell (Fc), serum dilution was optimized, and an appropriate sample diluent was selected. The sensor exhibited a detection range of 25-3200 ng mL-1. Intra-day and inter-day precision showed a coefficient of variation (CV) below 15% and an accuracy ranging between 85% and 115%. Finally, the biosensor was successfully applied to 15 clinical serum samples, showing significant correlation with the ELISA results. Compared to traditional methods, the SPR biosensor offers simpler preparation, faster analysis, and smaller sample volumes, which provides a new option for the TDM of bevacizumab.

The development of programmed death 1 (PD-1) checkpoint/programmed death-ligand 1 (PD-L1) interaction inhibitors has opened a new front in the treatment of carcinoma. An increasing amount of research is devoted to small-molecule compounds that target this PD-1/PD-L1 interaction. In this article, we report the discovery of three new PD-1/PD-L1 inhibitors from Scutellaria baicalensis Georgi and Sophora flavescens Aiton herbal extracts, namely baicalin, maackiain, and oxysophocarpine, using a comprehensive ligand fishing system, which integrates a dual-target surface plasmon resonance biosensor and a magnetic beads method. These three compounds were also confirmed in the serum in vivo, validated by binding affinity evaluation, molecular docking, and competitive enzyme-linked immunosorbent assay (ELISA) assay to act upon the interface of PD-1/PD-L1. Thus, these three ingredients could be potential PD-1/PD-L1 inhibitors and may serve as hit compounds for immunotherapeutic drug discovery. These results also highlight the efficiency of the dual-target surface plasmon resonance (SPR) and magnetic beads ligand fishing system in drug screening for disease treatment.

Second near‐infrared (NIR‐II) fluorescence imaging has emerged as a breakthrough technology for accurately revealing complex mechanisms in vivo owing to its high sensitivity, deeper tissue penetration, high spatiotemporal resolution, and high throughput. This review provides a comprehensive overview of NIR‐II fluorescence imaging, specifically focusing on the materials used, including single‐walled carbon nanotubes (SWCNTs), quantum dots (QDs), rare‐earth nanoparticles (RENPs), and organic fluorophores (OFs). It details their development, application, and advantageous performance in NIR‐II fluorescence imaging. Furthermore, this review highlights an approach to dynamic multiplexed NIR‐II fluorescence imaging in vivo that enables multitarget detection, providing a powerful tool for accurately and effectively assessing pathological processes and revealing complex biological mechanisms in vivo. Finally, it explores the aspects of translational medicine for NIR‐II imaging, addressing challenges, and future prospects related to material development, detection equipment, and unmet biomedical applications.

Houttuynia cordata Thunb., also known as Yuxingcao in Chinese, occupies a pivotal role in Asian traditional medicine and cuisine. The aerial parts and underground stems of H. cordata exhibit remarkable chemical diversity, particularly in essential oil. Nevertheless, the mechanisms regulating essential oil biosynthesis in H. cordata remain unclear. In this study, we present a quantitative overview of the proteomes across four tissues (flower, stem, leaf, and underground stem) of H. cordata, achieved through the application of the isobaric tag for relative and absolute quantitation (iTRAQ). Our research findings indicate that certain crucial ribosomal proteins and their interactions may significantly impact the production of essential oils in H. cordata. These results offer novel insights into the roles of ribosomal proteins and their associations in essential oil biosynthesis across various organisms of H. cordata.

D-dimer is a protein fragment generated during the fibrin breakdown by plasmin, and it serves as a mature biomarker for diagnosing thrombotic disorders. A novel immunoassay method based on surface plasmon resonance (SPR) has been developed, validated, and successfully applied for the quantification of D-dimer in human plasma with high sensitivity and rapidity. In this methodological study, we investigated the activity and stability of the SPR biosensor, sample pre-processing, washing conditions, intra-day and inter-day precision and accuracy and detection parameters, including a limit of detection of 8.3 ng/mL, a detection range spanning from 31.25 to 4000 ng/mL, and a detection time of 20 min. We compared D-dimer plasma concentration determination results using SPR with a classical latex-enhanced immunoturbidimetric immunoassay in 29 healthy individuals and thrombotic patients, and both methods exhibited consistency. Furthermore, we propose a hypothesis about the relationship between the concentration of D-dimer and its molecular weight. With an increase in the D-dimer concentration in plasma, the D-dimer approaches its simplest form (190 kDa).

Scutellaria baicalensis Georgi, also known as huang-qin in traditional Chinese medicine, is a widely used herbal remedy due to its anticancer, antivirus, and hepatoprotective properties. The S. baicalensis genome was sequenced many years ago; by contrast, the proteome as the executer of most biological processes of S. baicalensis in the aerial parts, as well as the secondary structure of the roots (xylem, phloem, and periderm), is far less comprehensively characterized. Here we attempt to depict the molecular landscape of the non-model plant S. baicalensis through a multi-omics approach, with the goal of constructing a highly informative and valuable reference dataset. Furthermore, we provide an in-depth characterization dissection to explain the two distinct flavonoid biosynthesis pathways that exist in the aerial parts and root, at the protein and phosphorylated protein levels. Our study provides detailed spatial proteomic and phosphoproteomic information in the context of secondary structures, with implications for the molecular profiling of secondary metabolite biosynthesis in non-model medicinal plants.

The rapid and accurate detection of illegal adulteration of chemical drugs into dietary supplements is a big challenge in the food chemistry field. Detection of compounds without a standard reference is even more difficult; however, this is a common situation. Here in this study, a novel "standard-free detection of adulteration" (SFDA) method was proposed and phosphodiesterase-5 inhibitor derivatives were used as an example to figure out the possibility and reliability of this SFDA method. After analysis by quadrupole coupled time of flight-tandem mass spectrometry detection and multivariable statistics, six common fragment ions were chosen to indicate whether adulteration was present or not, while 20 characteristic fragment ions indicated whether adulteration was by nitrogen-containing heterocycles or by anilines. Furthermore, the quantitative methods were conducted by high-performance liquid chromatography-tandem mass spectrometry. In a word, this strategy allows for a quick determination of dietary supplement adulteration without any need for standard materials, improving the efficacy of food safety testing.

Ethnopharmacological relevanceTanacetum parthenium (L.) Schultz-Bip, commonly known as feverfew, has been traditionally used to treat fever, migraines, rheumatoid arthritis, and cancer. Parthenolide (PTL), the main bioactive ingredient isolated from the shoots of feverfew, is a sesquiterpene lactone with anti-inflammatory and antitumor properties. Previous studies showed that PTL exerts anticancer activity in various cancers, including hepatoma, cholangiocarcinoma, acute myeloid leukemia, breast, prostate, and colorectal cancer. However, the metabolic mechanism underlying the anticancer effect of PTL remains poorly understood.Aim of the studyTo explore the anticancer activity and underlying mechanism of PTL in human cholangiocarcinoma cells.Material and methodsIn this investigation, the effects and mechanisms of PTL on human cholangiocarcinoma cells were investigated via a liquid chromatography/mass spectrometry (LC/MS)-based metabolomics approach. First, cell proliferation and apoptosis were evaluated using cell counting kit-8 (CCK-8), flow cytometry analysis, and western blotting. Then, LC/MS-based metabolic profiling along with orthogonal partial least-squares discriminant analysis (OPLS-DA) has been constructed to distinguish the metabolic changes between the negative control group and the PTL-treated group in TFK1 cells. Next, enzyme-linked immunosorbent assay (ELISA) was applied to investigate the changes of metabolic enzymes associated with significantly alerted metabolites. Finally, the metabolic network related to key metabolic enzymes, metabolites, and metabolic pathways was established using MetaboAnalyst 5.0 and Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway Database.ResultsPTL treatment could induce the proliferation inhibition and apoptosis of TFK1 in a concentration-dependent manner. Forty-three potential biomarkers associated with the antitumor effect of PTL were identified, which primarily related to glutamine and glutamate metabolism, alanine, aspartate and glutamate metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism, arginine biosynthesis, arginine and proline metabolism, glutathione metabolism, nicotinate and nicotinamide metabolism, pyrimidine metabolism, fatty acid metabolism, phospholipid catabolism, and sphingolipid metabolism. Pathway analysis of upstream and downstream metabolites, we found three key metabolic enzymes, including glutaminase (GLS), γ-glutamyl transpeptidase (GGT), and carnitine palmitoyltransferase 1 (CPT1), which mainly involved in glutamine and glutamate metabolism, glutathione metabolism, and fatty acid metabolism. The changes of metabolic enzymes associated with significantly alerted metabolites were consistent with the levels of metabolites, and the metabolic network related to key metabolic enzymes, metabolites, and metabolic pathways was established. PTL may exert its antitumor effect against cholangiocarcinoma by disturbing metabolic pathways. Furthermore, we selected two positive control agents that are considered as first-line chemotherapy standards in cholangiocarcinoma therapy to verify the reliability and accuracy of our metabolomic study on PTL.ConclusionThis research enhanced our comprehension of the metabolic profiling and mechanism of PTL treatment on cholangiocarcinoma cells, which provided some references for further research into the anti-cancer mechanisms of other drugs.

Traditional Chinese Medicine (TCM) is a supremely valuable resource for the development of drug discovery. Few methods are capable of hunting for potential molecule ligands from TCM towards more than one single protein target. In this study, a novel dual-target surface plasmon resonance (SPR) biosensor was developed to perform targeted compound screening of two key proteins involved in the cellular invasion process of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2): the spike (S) protein receptor binding domain (RBD) and the angiotensin-converting enzyme 2 (ACE2). The screening and identification of active compounds from six Chinese herbs were conducted taking into consideration the multi-component and multi-target nature of Traditional Chinese Medicine (TCM). Puerarin from Radix Puerariae Lobatae was discovered to exhibit specific binding affinity to both S protein RBD and ACE2. The results highlight the efficiency of the dual-target SPR system in drug screening and provide a novel approach for exploring the targeted mechanisms of active components from Chinese herbs for disease treatment.