Xiao-Shan Zheng

We evaluated the relationship between Raman spectral profiles obtained by single-cell Raman spectroscopy (SCRS) and autoinducer-2 (AI-2)-associated quorum sensing (QS) functional phenotypes using experimentally measured relative BB170 reporter responses as functional reference values. Based on this relationship, this study provides a rapid, label-free SCRS-based model for characterizing AI-2-associated QS functional phenotypes in bacterial isolates. The optimized residual neural network (ResNet) achieved an R2 of 0.8351 under spectrum-level 10-fold cross-validation, while strain-level aggregated evaluation showed good agreement with experimentally measured responses within the represented dataset (R2 = 0.8840). In an independent-isolates set of 21 strains from multiple sources spanning the No-, Low-, Medium-, and High-response categories, the model achieved a mean absolute error (MAE) of 0.092, a root mean squared error (RMSE) of 0.148, an R2 of 0.805, and an overall category accuracy of 80.95%. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) detected luxS expression in all 21 independent isolates, providing complementary evidence for their potential AI-2-associated signaling capacity. In addition, furanone C-30 treatment of W37 resulted in concordant decreases in experimentally measured and model-estimated relative BB170 reporter responses, further supporting the responsiveness of the captured spectral phenotype to within-strain functional perturbation. Integrated Gradients (IG) analysis highlighted coordinated Raman spectral regions associated with proteins, lipids, polysaccharides, nucleic acids, and sulfur-containing groups, suggesting that model estimates were based on multiband spectral patterns rather than a single characteristic feature.

Xihuang Pill (XHP), a classic traditional Chinese medicine (TCM) compound formula, exhibits antitumor activity, yet its therapeutic potential and underlying molecular mechanism against lung adenocarcinoma remain elusive. In this study, single-cell Raman spectroscopy (SCRS) was employed to investigate the effects and molecular mechanisms of XHP on human lung adenocarcinoma A549 cells. Results demonstrated that XHP inhibited the metabolic activity of A549 cells in a concentration-dependent manner, impaired cellular integrity, and induced structural remodeling of lipids and proteins accompanied by characteristic spectral shifts. Collectively, these alterations led to metabolic reprogramming in tumor cells. Pharmacological prediction and experimental validation indicated that prostaglandin-endoperoxide synthase 2 (PTGS2) is a relevant target mediating the pharmacological effects of XHP: XHP downregulated the expression of PTGS2, while overexpression of PTGS2 attenuated the therapeutic effects of XHP. This study provides direct, single-cell-level spectroscopic evidence for the efficacy of XHP against lung adenocarcinoma and reveals a PTGS2-associated mechanism, offering new insights for the development of TCM-based therapies for lung adenocarcinoma

Antifungal susceptibility test (AFST) is essential for guiding effective therapy, but it remains challenged by long turnaround times and the limited generalizability of single-parameter phenotypic readouts across diverse fungal species and antifungal classes. Here, we present a multifeature AFST (MAFST) platform that integrates single-cell Raman spectroscopy and brightfield imaging to enable rapid and pleiotropic assessment of antifungal responses. By unifying complementary metabolic and morphological features into a composite inhibition index (CII), MAFST captures coordinated cellular responses to antifungal exposure that cannot be reliably resolved by individual metrics alone. Benchmarking against the broth microdilution demonstrates that CII-based MAFST achieves high concordance across multiple Candida species and antifungal classes while substantially reducing the time required for susceptibility determination. Thus, by capturing single-cell morphological, developmental, and metabolic drug responses in a fast, accurate, and widely applicable fashion, MAFST can contribute to tackling fungal infections.

Antibacterial agents, particularly surfactants, play crucial roles in the personal and home care industries. However, current methods for assessing their efficacy and mechanism are commonly time-consuming and expensive. Here, we established a ramanome-based approach to investigate the antibacterial mechanisms of cationic and anionic surfactants with varying alkyl chain lengths against Staphylococcus aureus as a model. Our findings further elucidate the synergy between anionic surfactants and acidic pH. Cell membrane integrity was disrupted by all of the surfactants, as revealed by the decrease in Raman bands assigned to major cellular components (nucleic acids, proteins, and cytochrome), leading to the leakage of cellular components. Moreover, the composition of the cell membrane was altered due to insertion of cationic surfactant, evidenced by the emergence of surfactant-characteristic bands in the spectrum of S. aureus; yet this was observed only under acidic conditions for anionic surfactants. Remarkably, changes in Raman bands of staphyloxanthin and S═O which are biomarkers of cellular oxidative states revealed that acidic conditions accelerated cell death induced by the anionic surfactant. These findings illustrate distinct mechanisms of action for cationic and anionic surfactants and suggest that ramanomics offers a rapid, low-cost, comprehensive, and mechanism-revealing approach for the assessment and screening of surfactants.

In Raman-activated Cell Ejection and Sequencing (RACE-Seq), success rate and sequence coverage have generally been low for shotgun sequencing of individual post-RACE cells. Here we quantitatively evaluated the influence of cell lysis condition, nucleic acid amplification condition, and parameters of Raman measurement on RACE-Seq performance. Variations in laser energy input during Raman signal acquisition, but not duration of alkaline lysate lysis, temperature, or measurement under dry or aqueous conditions, are vital to the success of multiple displacement amplification (MDA). In fact, laser irradiation is reversely linked to MDA product quality. However, introduction of oils prior to MDA, by mitigating such negative effects of Raman irradiation, elevates genome coverage of post-RACE E. coli cells from <20% to ~50%, while greatly improves the success rate of RACE-Seq for soil microbiota. Our findings provide a practical solution for enhancing RACE-Seq performance, and pinpoint protection of cells from laser irradiation as priority in method development.

Intracellular pH is an important modulator of cell functions and its subtle change may dramatically affect the cellular activities and cause diseases. A reliable imaging of the intracellular pH is still a great challenge. We imaged the intracellular pH during the cell cycle at the single living cell level using newly designed cell-penetrating peptide conjugated pH nanosensors on a home-built in situ microscopic cell culture platform. The conjugated cell-penetrating peptide greatly enhanced the uptake of nanosensors without sacrificing the pH response. We observed a gradual alkalization from interphase to prophase and rapid acidification from prometaphase to telophase, reflecting variation and consumption of the species related to the energy storage during cell cycle. We realized SERS-based pH and fluorescence dual-mode imaging when the pH sensor was further modified with fluorescence dye. The integration of SERS imaging with in situ microscopic cell culture system offers great opportunity for revealing the intracellular pH-related biological and pathological processes.

Surface-enhanced Raman spectroscopy (SERS) inherits the rich chemical fingerprint information on Raman spectroscopy and gains sensitivity by plasmon-enhanced excitation and scattering. In particular, most Raman peaks have a narrow width suitable for multiplex analysis, and the measurements can be conveniently made under ambient and aqueous conditions. These merits make SERS a very promising technique for studying complex biological systems, and SERS has attracted increasing interest in biorelated analysis. However, there are still great challenges that need to be addressed until it can be widely accepted by the biorelated communities, answer interesting biological questions, and solve fatal clinical problems. SERS applications in bioanalysis involve the complex interactions of plasmonic nanomaterials with biological systems and their environments. The reliability becomes the key issue of bioanalytical SERS in order to extract meaningful information from SERS data. This review provides a comprehensive overview of bioanalytical SERS with the main focus on the reliability issue. We first introduce the mechanism of SERS to guide the design of reliable SERS experiments with high detection sensitivity. We then introduce the current understanding of the interaction of nanomaterials with biological systems, mainly living cells, to guide the design of functionalized SERS nanoparticles for target detection. We further introduce the current status of label-free (direct) and labeled (indirect) SERS detections, for systems from biomolecules, to pathogens, to living cells, and we discuss the potential interferences from experimental design, measurement conditions, and data analysis. In the end, we give an outlook of the key challenges in bioanalytical SERS, including reproducibility, sensitivity, and spatial and time resolution.

We describe a facile and low-cost approach for a flexibly integrated surface-enhanced Raman scattering (SERS) substrate in microfluidic chips. Briefly, a SERS substrate was fabricated by the electrostatic assembling of gold nanoparticles, and shaped into designed patterns by subsequent lift-up soft lithography. The SERS micro-pattern could be further integrated within microfluidic channels conveniently. The resulting microfluidic SERS chip allowed ultrasensitive in situ SERS monitoring from the transparent glass window. With its advantages in simplicity, functionality and cost-effectiveness, this method could be readily expanded into optical microfluidic fabrication for biochemical applications.