Recent studies have demonstrated that terahertz spectroscopy can identify starch absorption peaks in solid pellets containing powdered plant samples with minimal interference of other plant matrices. Here, we applied this analytical method to assess photosynthetic capacity in plant leaves with a relatively low starch content. To monitor starch produced through photosynthesis, rice leaves were measured in the frequency range of 3.0-9.5 THz using a Fourier-transform spectrometer equipped with a bolometer. A starch over-accumulation mutant (α-glucan water dikinase1) and wild type (cv. Nipponbare) were harvested after 2 and 3 months of cultivation. In the terahertz measurements, all leaves exhibited an absorption peak at 9.0 THz attributed to starch and the peak intensities were consistent with starch contents obtained by chemical analysis. Furthermore, correlations were found between second derivative signals and starch content (R[2] = 0.86-0.87). However, a higher accuracy model was developed using peak height calculated using the peak top at 9.0 THz and a baseline between 8.65 and 9.35 THz (R[2] = 0.91). These results indicate that just three frequencies are sufficient on behalf of the full range spectra, suggesting the feasibility of a starch sensor for future applications. Therefore, terahertz technology can serve as a valuable analytical method for assessing starch accumulation and photosynthetic capacity in unpurified plant leaves.

Molecular absorption spectroscopy provides a species-selective basis for quantitative gas sensing and compact optical diagnostics in combustion, industrial processes, and emission monitoring. However, in scanned semiconductor laser spectrometers operating without an online frequency reference, residual tuning nonlinearities introduce deviations in the wavenumber coordinate that affect spectral registration, baseline variation, and concentration retrieval. Here, we report a joint inversion method for CO2 direct absorption spectroscopy, in which the molecular absorption-band structure is used as an internal spectral constraint for effective wavenumber coordinate correction and concentration retrieval. Using the prior trajectory derived from the scan as the initial coordinate, the method estimates the residual wavenumber correction for each scan, a compact model of the non-absorbing intensity baseline, and the CO2 mole fraction within a unified forward model, without requiring an online Fabry-Perot etalon, reference absorption cell, or wavemeter. In experiments with a DFB laser operating near 4990 cm[-1], a 45.0 cm effective path length, and standard CO2/N2 gas mixtures with concentrations ranging from 1000 to 4500 ppm, the absorption-band prior method reduced the absorbance residual RMSE from 2.01 × 10[-3] to 9.60 × 10[-4] compared with that of single-peak retrieval. For concentration retrieval, the mean absolute relative error and concentration standard deviation decreased from 4.08% to 1.63% and from 92.3 ppm to 38.9 ppm for single-scan retrieval and from 2.32% to 1.39% and from 60.8 ppm to 27.1 ppm after averaging over nine scan periods, respectively. These results show that spectroscopic constraints applied across the absorption band provide an effective approach for improving spectral registration, concentration accuracy, and retrieval stability over short measurement intervals in DAS systems operating without an online frequency reference.

Jerantinines are natural indole alkaloids with promising anticancer activity in breast cancer cells via mechanisms of action involving tubulin binding and disruption of microtubule dynamics, with growth inhibition values, GI50 < 0.4 µM. However, metabolic responses of different breast cancer phenotypes are not yet understood. In this work, LC-MS-based untargeted metabolomics was used to investigate the response of four selected breast cancer cell lines representing different subtypes (MCF-7, BT-474, MDA-MB-231, and SKBR-3) to treatment with Jerantinine A acetate (JAa). Based on univariate, multivariate and pathway analyses, the results showed metabolic perturbations in amino-acid metabolism, fatty acid oxidation, and redox homeostasis. In breast cancer cells treated with 0.4 µM of JAa for 72 h, the elevated levels of oxidation-related metabolites, including 2-oxoglutarate, butyryl carnitine, propionyl carnitine, and 5-oxo-L-proline, indicated increased oxidative stress. Interestingly, only MCF-7 cells accumulated the antioxidants glutathione and taurine relative to untreated controls, and this response was accompanied by upregulation of wild-type p53 in this cell line. This p53-associated antioxidant capacity offers a mechanistic explanation for the difference in observed GI50 values: GI50 of 0.37 µM in MCF-7 compared to 0.19 µM and 0.14 µM in SKBR-3 and BT-474 cells, respectively, both of which harbour mutant p53 and did not mount an equivalent antioxidant response. These findings provide new insights into the metabolic effects of JAa on breast cancer cells and further support its potential as an anticancer agent. The observed metabolic differences between cell lines may inform future strategies for personalized therapy.

Biomass-based CDs constitute a rapidly developing, eco-friendly category of nanomaterials engineered mainly for ecological biosensing applications. By utilizing organic materials, including crop residues, botanical wastes and discarded food products, as the carbon source or precursor, these CDs offer a green synthesis method that avoids the risk of traditional chemical synthesis. As these green CDs are fabricated from natural precursors, these nanomaterials may contain surface-bound heteroatoms, like sulfur, nitrogen, and phosphorus, derived from their organic feedstocks. Therefore, these functionalized CDs facilitate the quick, easy and highly sensitive identification of environmental pollutants, such as pharmaceutical residues, toxic heavy metals, agricultural pesticides and novel contaminants. The presence of heteroatoms improves their fluorescence properties, selectivity, and interaction with target analytes. Consequently, these CDs exhibit rapid, user-friendly, and sensitive detection of environmental contaminants, including pesticides, heavy metals, antibiotics, and emerging pollutants. From this perspective, green-synthesised CDs are expected to have a substantial long-term impact on environmental monitoring. Their compatibility with portable sensing platforms, suitability for field-deployable platforms, and ability to detect contaminants at trace levels make them promising candidates for next-generation environmental monitoring systems. However, the transformation of these sustainable nanomaterials into practical environmental solutions will require further investigations into their biomass precursors, structural property correlations, and mechanism-driven sensing strategies. Instead of mainly focusing on lower detection limits, future research studies should focus on mechanistic insights, real environmental stability and real-world applicability to enable the practical utilisation of sustainable carbon dot sensing platforms.

Understanding the behaviour of lipids during digestion of food and formulations can enable more rational design of lipid-based materials to enhance the oral delivery of drugs and nutrients. Traditionally, light microscopy and electron microscopy have been used to 'watch' digestion at the droplet level, while small angle X-ray scattering has more recently given structural insights at the ensemble level, however none of these techniques provide information on local changes in chemical composition of a digesting fat droplet. In this study, we used synchrotron Fourier transform infrared microspectroscopy (s-FTIRM) as a chemical imaging technique to map the distributions of milk components and their digestion products in individual fat globules to obtain a better understanding of the interfacial characteristics and chemical specificity behind the structural changes that occur during digestion. The results showed localisation of fatty acids at the core and the oil/water interface of digesting fat globules and the presence of fatty acid-calcium soaps, which exist both as isolated aggregates and are partially accumulated at the interface. This proof-of-concept study highlights the novel application of s-FTIRM as a label-free, chemically-specific imaging tool to resolve the spatial distribution of digestion products, such as free fatty acids and fatty acid-calcium soaps, within and around individual milk fat globules. This approach provides new insights into the localised chemical changes during lipid digestion at the single droplet level.

The development of diagnostic tests that offer rapid and quantitative results can improve the health of patients, suffering from both chronic and acute diseases. Inflammatory bowel disease (IBD) is a chronic autoimmune condition, where a patient can undergo periods of sudden flare-ups and prolonged remission. The current biomarkers of interest are either detected using central laboratory methods, or, are non-specific to IBD. In this work, we develop a series of lateral flow immunoassays for the detection of IBD biomarkers lactoferrin and myeloperoxidase, in an artificial stool matrix. By utilising surface enhanced Raman scattering (SERS) active nanotags, we demonstrate the feasibility of these SERS-based lateral flow immunoassays for proof-of-concept singleplex and duplex detection via Raman mapping. Due to the variation in background caused by the stool matrix, we have investigated methods of assessing the SERS signal from the Raman maps, to transition the tests from giving qualitative to quantitative results. This has been achieved by extracting the SERS intensities from the maps associated with non-specific interactions, to define unique intensity thresholds for each lateral flow immunoassay, overall improving the sensitivity, reproducibility, and linearity in limit of detection studies. We achieved limits of detection of 3.2 ng mL[-1] for lactoferrin and 5.0 ng mL[-1] for myeloperoxidase, and limits of quantification of 18.8 ng mL[-1] for lactoferrin and 8.0 ng mL[-1] for myeloperoxidase. These values are well below the "normal" level of these biomarkers in stool, indicating this assay would be able to accurately monitor changing biomarker concentrations. Furthermore, we used a ratiometric approach to evaluate the maps from duplex lateral flows and detect each biomarker simultaneously on the same strip. Overall, this work demonstrates that it is possible to quantitatively detect IBD biomarkers in stool on a lateral flow with SERS, using improved Raman signal analysis methods to overcome complex sample background interference and enhance sensitivity.

The development of fluorescent probes that integrate spatiotemporal organelle targeting with analyte sensing remains a significant challenge. Herein, we report a modular library of four styryl pyridinium fluorophores (Py-Cz-CN, Py-Cz, Py-Am-CN, and Py-Am) based on a donor-π-acceptor (D-π-A) architecture for sequential organelle targeting and sulfite sensing. All four probes exhibited rapid and selective plasma membrane localization, with high colocalization coefficients (PCC = 0.81-0.88). Notably, Py-Cz underwent spontaneous time-dependent translocation from the plasma membrane to mitochondria over 6 h, with the Pearson's correlation coefficient increasing from 0.15 to 0.87, and this mitochondrial accumulation was confirmed to be ΔΨm-dependent by CCCP-induced membrane depolarization. In contrast, cyano substitution at the vinyl bridge preserved plasma membrane localization while introducing a Michael acceptor for selective reaction-based fluorescence turn-off sensing of bisulfite/sulfite. Py-Cz-CN exhibited high sensitivity toward bisulfite (LOD = 5.9 μM) and enabled both exogenous and endogenous sulfite imaging in live cells. DFT and TDDFT calculations further supported the excited-state characteristics and sensing mechanism. This work establishes a simple molecular design strategy for combining organelle-specific trafficking with analyte-responsive fluorescence in multifunctional small-molecule probes.

The toxicity of organophosphate (OP) insecticides and pesticides is increasing in the human population owing to their indiscriminate use globally. The need to achieve food security for the rising population is the main reason for the excessive use of such chemical compounds. The hazardous effects of OP pesticides can be mitigated only through conscientious efforts in detecting them in various food and water resources. In the present study, we developed carbon quantum dots (CQDs) and recombinant Nus-organophosphorus hydrolase (Nus-OPH) enzyme co-immobilized chitosan polymer film-based biosensors for the detection of OP compounds, such as coumaphos (CP), methyl parathion (MP) and ethyl paraoxon (EP), present in river water and household water resources using a fiber optic spectrophotometer. The E. coli strains were utilized for the expression of the Nus-OPH enzyme, purified and subsequently immobilized on the chitosan thin films containing CQDs that were synthesized from triethylene diamine and phthalic acid through microwave irradiation. The thin-film biosensors composed of CQDs-Nus-OPH were characterized utilizing several methods like XRD, XPS, TEM, CLSM, and enzyme activity measurements. The biosensing studies of CP, EP, and MP were performed by recording fluorescence changes using reflectance probe-mediated measurements in a fiber optic spectrophotometer. This study determined that the CQDs-Nus-OPH thin film-based biosensor has the potential to detect/analyze EP, CP, and MP in the 0-100 µM range with the limits of detection of 145 ppb, 178 ppb, and 188 ppb, respectively, within a time frame of 5 minutes. The accuracy of the biosensor in the OP-spiked water matrices was found to be 100%-102%, indicating that the CQDs-Nus-OPH thin-film biosensors can detect the analyte accurately. These results indicate that this biosensor platform shows potential for organophosphate detection, although further studies are needed to improve specificity and analyte discrimination.

Urea is an important clinical biomarker for health monitoring and is also used as a nitrogen-containing adulterant in food products to artificially increase the apparent protein content, creating an important demand for portable and reliable detection methods. In this work, Cu2O nanocubes were used as representative copper-based nanozymes to construct a dual-channel colorimetric platform for urea sensing by combining UV-vis absorbance with smartphone-assisted RGB analysis. In the sensing cascade, urease first catalyzes the hydrolysis of urea to generate NH3, leading to an increase in solution pH. Since the peroxidase-like (POD-like) activity of Cu2O is highly pH-dependent, this pH evolution is converted into a concentration-dependent colorimetric response through the H2O2-mediated oxidation of 3,3',5,5'-tetramethylbenzidine (TMB). However, residual urease can continue to hydrolyze urea during the subsequent color-development step, causing further pH drift and unstable optical signals. Importantly, Cu2O nanocubes function not only as catalytic nanozymes for TMB oxidation, but also as reaction terminators that suppress the residual urease activity, thereby stabilizing the system pH and improving the signal reliability. Based on this mechanism, a gelatin hydrogel sensor was further developed for portable urea monitoring. The absorbance channel showed a linear response over 0-2.2 mM with a detection limit of 5.75 μM, while the RGB channel provided smartphone-readable quantitative analysis. The proposed platform was successfully applied to urea determination in sweat and milk samples, demonstrating its potential for portable biosensing and food safety analysis.

Lower respiratory tract infections (LRTIs) remain a leading cause of mortality worldwide. Rapid and comprehensive etiological diagnosis is critical yet challenging: existing approaches, from culture to multiplex PCR and mNGS, carry distinct limitations in one or more of turnaround time, sensitivity, cost, or quantitative resolution. We developed a 33-plex digital PCR (dPCR) assay for absolute quantification of bacteria, viruses, fungi, and atypical pathogens in bronchoalveolar lavage fluid (BALF). Systematic evaluation of five liquefaction protocols identified dithiothreitol (DTT) as the optimal universal pretreatment, ensuring balanced RNA and DNA recovery. The assay demonstrated reliable quantification from 10 to 10[5] copies per test for all 33 targets (R[2] > 0.99), with 27 targets showing linearity across full range and 6 targets exhibiting linearity from 10 to 10[4] copies per test (10[5] as upper detection limit). The limit of detection was 5 copies per test for all targets. In 105 BALF samples, dPCR achieved a significantly higher positive rate than culture (96.19% vs. 83.81%, p < 0.05), particularly for polymicrobial infections (85.71% vs. 40.00%). Concordance with mNGS for primary pathogens was 86.96% (20/23) for 14 primary pathogens across 23 positive samples. The complete workflow from sample pre-treatment to results required only 2.5 hours. Serial BALF dPCR quantification of P. aeruginosa and K. pneumoniae in one severe LRTI case tracked pathogen loads in parallel with clinical course and antibiotic responses, preliminarily supporting the technical feasibility of dynamic monitoring. This study demonstrates the feasibility of a rapid 33-plex dPCR assay for comprehensive respiratory pathogen profiling in BALF, with potential for dynamic monitoring in critically ill patients.

Surface-enhanced Raman scattering (SERS) enables real-time analysis of volatile organic compounds (VOCs) and other gaseous molecules using portable Raman devices, offering faster measurements compared to GC-MS. To facilitate this, numerous solid SERS substrates have been developed and applied across multiple fields. However, they often require complex fabrication due to poor adsorption of gases onto solid surfaces leading to high production costs and inconsistencies in sensitivity and reproducibility. In contrast, colloidal nanoparticles are less expensive to produce and established facile synthesis methods help their broader implementation. In this study a simple approach has been conceptualised whereby gaseous analytes are bubbled directly into colloidal nanoparticles. This was achieved by pumping air into a flask containing the volatile analyte, which in turn was bubbled into a vial containing colloidal nanoparticles and measured with a portable Raman spectrometer. Initially, 3-mercapto-hexanol (3MH) was successfully measured using this method. 3MH was chosen due to the method originally being developed to measure thiols associated to underarm odour. Subsequently, 3MH was used to optimize the air flow rate by testing at eight different flow rates. The results demonstrated that flow rate influenced the timing of analyte spectral appearance as seen by the rate of change in peak height at 634 cm[-1] for 3MH, with the most reproducible results observed at 50 mL min[-1]. The technique was further evaluated with two other analytes: 2-methyl-3-mercapto-pentanol (2M3MP) and indole. The latter needed the addition of an aggregating agent (NaCl) to the nanoparticles. These findings indicate that this approach offers a promising alternative for detecting gaseous analytes using colloidal nanoparticles.

The misuse of antibiotics in the livestock industry and their improper discharge into natural water bodies threaten public health and ecological security. The sensitive and reliable detection of antibiotic pollution is crucial for preventing the associated damage. In this work, we proposed a MoS2@Ga-doped ZnO heterojunction-based surface-enhanced Raman scattering platform for antibiotic detection. The heterostructure facilitates the separation and transfer of photogenerated electron-hole pairs while suppressing their recombination. This enhances the interfacial charge transfer between the heterojunction and the target molecule, leading to a significant amplification of the molecular Raman signal. A simple mixing of the sample with the heterojunction enables highly sensitive detection of antibiotics. The sensing performance was evaluated by detecting tetracycline in water and milk, with limits of detection of 1.82 × 10[-11] mol L[-1] and 2.51 × 10[-12] mol L[-1] in these two matrices, respectively. Furthermore, this platform exhibited robust practicality, anti-interference capability and stability. These advantages highlight the platform's potential for accurate monitoring of antibiotics in different matrices.

Parathyroid hormone (PTH) is essential for calcium-phosphate homeostasis and bone metabolism. Abnormal PTH levels are closely associated with chronic kidney disease and several types of cancer. Current mainstream immunological PTH detection methods cannot achieve single-cell analysis, and dual-signal detection strategies are scarce. Herein, we designed a dual-signal nano-microelectrode for single-cell PTH detection by modifying gold-deposited nanopipettes with ferrocene (Fc)-labeled DNA-complementary DNA double strands and 6-mercaptohexanol (6-MCH). Under optimized conditions, square-wave voltammetry (SWV) showed a linear response to PTH in the range of 1-500 pg mL[-1] (R[2] = 0.99), and EIS exhibited linearity in the range of 1-1000 pg mL[-1] (R[2] = 0.98). The sensor demonstrated high selectivity against common intracellular interferents and good electrode-to-electrode consistency. Using three-dimensional micro-manipulation technology, minimally invasive detection of individual cells was achieved, and significant changes in electrochemical signals before and after cell penetration were revealed. Furthermore, the intracellular PTH concentration gradually decreased with prolonged extracellular Ca[2+] incubation, consistent with the regulatory mechanism of calcium-sensing receptors. This dual-mode nano-micro electrode is a new type of tool that enables rapid, low-cost, label-free and highly specific single-cell PTH analysis.

Determining the higher order structure (HOS) of proteins and protein complexes is central to understanding their functions, dynamics, and interactions. Traditional structural biology approaches, such as X-ray crystallography and nuclear magnetic resonance, provide high-resolution snapshots but often require large amounts of homogeneous samples and may miss dynamic or heterogeneous states. Mass spectrometry (MS) has become an indispensable tool for sensitive and rapid analysis of intact proteins and assemblies under native or near-native conditions. This review discusses the major MS-based strategies for probing HOS. Native mass spectrometry (nMS) preserves non-covalent interactions and exhibits characteristic charge-state distributions that report on folding, while native top-down fragmentation and ion mobility spectrometry provide sequence-specific and conformation-specific information. Hydrogen-deuterium exchange MS measures backbone amide exchange rates to map regions of solvent accessibility, ligand binding, and allosteric regulation in solution. Covalent labeling MS irreversibly modifies solvent-accessible side chains, allowing epitope mapping and detection of subtle conformational changes, while fast photochemical oxidation of proteins offers microsecond snapshots of transient structures. Chemical cross-linking MS applies bifunctional reagents to capture proximity between residues or subunits, providing distance restraints for integrative modeling and proteome-wide interaction mapping. We outline recent advances in instrumentation, software, labeling chemistry and in-cell techniques across these modalities, and we illustrate their applications to characterizing membrane proteins, large assemblies, therapeutic antibodies, intrinsically disordered proteins, and protein-ligand complexes. Together, these tools offer complementary insights into HOS that are reshaping structural biology, biopharmaceutical development and mechanistic studies.

A biomimetic thin organic film electrode (TOFE) electrochemical platform was developed to investigate the interfacial behavior of vinblastine (VBL), a widely used anticancer vinca alkaloid. The system was constructed by immobilizing an organic phase (containing nitrobenzene, decamethylferrocene (DmFc) and a perchlorate-based supporting electrolyte) onto an edge-plane pyrolytic graphite electrode, which, when immersed in an aqueous phase (containing an inorganic perchlorate salt), collectively formed a stable solid|organic|aqueous interface. Although VBL did not exhibit a direct electrochemical response within the TOFE system, its presence significantly altered the voltammetric behavior of the DmFc redox mediator, leading to a marked decrease in DmFc peak currents and enabling indirect analysis. Frequency- and amplitude-dependent square wave voltammetry (SWV) analyses revealed a transition toward a kinetically hindered regime, consistent with VBL adsorption and accumulation at the liquid|liquid interface. The TOFE platform enabled indirect determination of VBL by SWV over the linear range of 5.0-100.0 µmol L[-1] with limits of detection of 1.62 µmol L[-1] and 1.65 µmol L[-1] for the voltammetric scan in anodic and cathodic directions, respectively. Comparison with previously investigated anticancer agents, vincristine and docetaxel, VBL revealed distinct variations in interfacial kinetic behavior, highlighting the ability of the TOFE system to distinguish between pharmaceutical compounds based on their interfacial properties. These findings confirm that TOFE-based electrochemistry provides a reliable approach for probing drug-liquid interface interactions and enables indirect detection of electrochemically inactive species, demonstrating its versatility for both mechanistic investigations and analytical applications in pharmaceutical chemistry.