Occlusion enhanced pan-cancer classification via deep learning.

Quantitative measurement of RNA expression levels through RNA-Seq is an ideal replacement for conventional cancer diagnosis via microscope examination. Currently, cancer-related RNA-Seq studies focus on two aspects: classifying the status and tissue of origin of a sample and discovering marker genes. Existing studies typically identify marker genes by statistically comparing healthy and cancer samples. However, this approach overlooks marker genes with low expression level differences and may be influenced by experimental results. This paper introduces "GENESO," a novel framework for pan-cancer classification and marker gene discovery using the occlusion method in conjunction with deep learning. we first trained a baseline deep LSTM neural network capable of distinguishing the origins and statuses of samples utilizing RNA-Seq data. Then, we propose a novel marker gene discovery method called "Symmetrical Occlusion (SO)". It collaborates with the baseline LSTM network, mimicking the "gain of function" and "loss of function" of genes to evaluate their importance in pan-cancer classification quantitatively. By identifying the genes of utmost importance, we then isolate them to train new neural networks, resulting in higher-performance LSTM models that utilize only a reduced set of highly relevant genes. The baseline neural network achieves an impressive validation accuracy of 96.59% in pan-cancer classification. With the help of SO, the accuracy of the second network reaches 98.30%, while using 67% fewer genes. Notably, our method excels in identifying marker genes that are not differentially expressed. Moreover, we assessed the feasibility of our method using single-cell RNA-Seq data, employing known marker genes as a validation test.

Long-read sequencing reveals extensive gut phageome structural variations driven by genetic exchange with bacterial hosts.

Genetic variations are instrumental for unraveling phage evolution and deciphering their functional implications. Here, we explore the underlying fine-scale genetic variations in the gut phageome, especially structural variations (SVs). By using virome-enriched long-read metagenomic sequencing across 91 individuals, we identified a total of 14,438 nonredundant phage SVs and revealed their prevalence within the human gut phageome. These SVs are mainly enriched in genes involved in recombination, DNA methylation, and antibiotic resistance. Notably, a substantial fraction of phage SV sequences share close homology with bacterial fragments, with most SVs enriched for horizontal gene transfer (HGT) mechanism. Further investigations showed that these SV sequences were genetic exchanged between specific phage-bacteria pairs, particularly between phages and their respective bacterial hosts. Temperate phages exhibit a higher frequency of genetic exchange with bacterial chromosomes and then virulent phages. Collectively, our findings provide insights into the genetic landscape of the human gut phageome.

Electrochemical Detection of Ammonia in Water Using NiCu Carbonate Hydroxide-Modified Carbon Cloth Electrodes: A Simple Sensing Method.

Excessive ammonia nitrogen can potentially compromise the safety of drinking water. Therefore, developing a rapid and simple detection method for ammonia nitrogen in drinking water is of great importance. Nickel-copper hydroxides exhibit strong catalytic capabilities and are widely applied in ammonia nitrogen oxidation. In this study, a self-supported electrode made of nickel-copper carbonate hydroxide was synthesized on a carbon cloth collector via a straightforward one-step hydrothermal method for rapid ammonia nitrogen detection in water. It exhibits sensitivities of 3.9 µA µM-1 cm-2 and 3.13 µA µM-1 cm-2 within linear ranges of 1 µM to 100 µM and 100 µM to 400 µM, respectively, using a simple and rapid i-t method. The detection limit is as low as 0.62 µM, highlighting its excellent anti-interference properties against various anions and cations. The methodology's simplicity and effectiveness suggest broad applicability in water quality monitoring and environmental protection, particularly due to its significant cost-effectiveness.

Defect monitoring method for Al-CFRTP UFSW based on BWO-VMD-HHT and ResNet.

Underwater friction stir welding (UFSW) achieves reliable joining between dissimilar materials and meets the welding demand for function and properties in lightweight structures of modern engineering. A defect monitoring method based on Variational Mode Decomposition optimized by Beluga Whale Optimization and Hilbert-Huang Transform (BWO-VMD-HHT) is proposed to solve the unclear feature of AE signal in UFSW due to the aqueous medium. UFSW experiments on Al alloy and carbon fiber reinforced thermoplastic (CFRTP) are carried out with AE signals measured. The time-frequency domain features of AE signals are extracted by BWO-VMD-HHT. The experimental results show that the main frequency of the AE signal is 22.5 kHz, and surface crack defects, shallow hole defects, and deep hole defects are accompanied by the transfer phenomena of different frequency components. Then, the feature vectors are built by frequency components in the BWO-VMD-HHT spectrum and reduced by principal component analysis, including 22.5 kHz, 24 kHz, 20.6 kHz, 18.4 kHz, 17.3 kHz, and 15.6 kHz. The feature vectors are divided into the train and test sets, and the welding defect prediction model (ResNet18-attention) is built by ResNet18 and trained by feature vectors. In the test set, the ResNet18-attention is compared with the BP, SVM, and RBF. Test results show that the precision of models has improved by at least 10%, which are trained by BWO-VMD-HHT features vector. Also, ResNet18-attention has achieved an average precision of 0.906 and recognizes the category of weld defect accurately, and this method can be applied to the defect monitoring of UFSW.

Highly active chromium-based selective ethylene tri-/tetramerization catalysts supported by alkenylphosphanyl PNP ligands.

A series of novel diphosphinoamine (PNP) ligands bearing a P-alkenyl group were synthesized and applied in chromium-catalyzed selective ethylene tri-/tetramerization by in situ combination of Cr(acac)3 and modified methylalumoxane (MMAO-3A). The ligand substitution and oligomerization conditions have a remarkable influence on the catalytic activity and controllable selectivity. Most of these PNP ligands are highly active for ethylene tri-/tetramerization with considerable selectivity. An asymmetric diisopropenylphosphanyl ligand with an N-cyclohexyl group achieved the highest activity of 2036 kg (g Cr h-1)-1 with a high total selectivity of 81.1 wt% toward valuable 1-hexene (43.0 wt%) and 1-octene (38.1 wt%) at 40 bar ethylene and 60 °C. An asymmetric mixed isopropenyl/ethylphosphanyl ligand with an N-isopropyl group exhibited a high 1-octene selectivity of 65.5 wt% and a high total 1-hexene/1-octene selectivity (91.5 wt%) with a high activity of 1256 kg (g Cr h-1)-1.

Fourier Raman light field microscopy based on surface-enhanced Raman scattering.

Raman scattering, as a vibrational spectrum that carries material information, has no photobleaching that enables long-duration imaging. Raman spectra have very narrow emission peaks, and multiplex Raman imaging can be achieved by using different Raman scattering peak signals. These advantages make Raman imaging widely used in biology, cytology, and medicine, which has a wider range of application scenarios. However, obtaining a three-dimensional (3D) Raman image requires scanning for tens of minutes to several hours at present. Therefore, a fast non-scanning 3D Raman imaging method is greatly needed. In this article, we propose a Fourier Raman light field microscopy based on surface-enhanced Raman scattering (sers-FRLFM). Using flower-like gap-enhanced Raman nanoparticles (F-GERNs) to enhance Raman scattering signals, a Fourier-configured light field microscope (LFM) is capable of recording complete four-dimensional Raman field information in a single frame, facilitating the 3D reconstruction of the Raman image without generating reconstruction artifacts at the native object plan. Moreover, F-GERNs can mark specific locations and have the potential to become a new tracing method to achieve specific imaging. This imaging method has great potential in the 3D real-time Raman imaging of cells, microorganisms, and tissues with the lateral resolution of 2.40 µm and an axial resolution of 4.02 µm.

Crossmodal to unimodal transfer of temporal perceptual learning.

Subsecond temporal processing is crucial for activities requiring precise timing. Here, we investigated perceptual learning of crossmodal (auditory-visual or visual-auditory) temporal interval discrimination (TID) and its impacts on unimodal (visual or auditory) TID performance. The research purpose was to test whether learning is based on a more abstract and conceptual representation of subsecond time, which would predict crossmodal to unimodal learning transfer. The experiments revealed that learning to discriminate a 200-ms crossmodal temporal interval, defined by a pair of visual and auditory stimuli, significantly reduced crossmodal TID thresholds. Moreover, the crossmodal TID training also minimized unimodal TID thresholds with a pair of visual or auditory stimuli at the same interval, even if crossmodal TID thresholds are multiple times higher than unimodal TID thresholds. Subsequent training on unimodal TID failed to reduce unimodal TID thresholds further. These results indicate that learning of high-threshold crossmodal TID tasks can benefit low-threshold unimodal temporal processing, which may be achieved through training-induced improvement of a conceptual representation of subsecond time in the brain.

Individual cell modification with cell surface specific atom transfer radical polymerization for enhanced Cr(VI) removal.

Modifying cells with polymers on the surface can enable them to gain or enhance function with various applications, wherein the atom transfer radical polymerization (ATRP) has garnered significant potential due to its biocompatibility. However, specifically initiating ATRP from the cell surface for in-situ modification remains challenging. This study established a bacterial surface-initiated ATRP method and further applied it for enhanced Cr(VI) removal. The cell surface specificity was facilely achieved by cell surface labelling with azide substrates, following alkynyl ATRP initiator specifically anchoring with azide-alkyne click chemistry. Then, the ATRP polymerization was initiated from the cell surface, and different polymers were successfully applied to in-situ modification. Further analysis revealed that the modification of Shewanella oneidensis with poly (4-vinyl pyridine) and sodium polymethacrylate improved the heavy metal tolerance and enhanced the Cr(VI) removal rate of 2.6 times from 0.088 h-1 to 0.314 h-1. This work provided a novel idea for bacterial surface modification and would extend the application of ATRP in bioremediation.

Cationic liposomes overcome neutralizing antibodies and enhance reovirus efficacy in ovarian cancer.

Reovirus (Reo) has shown promising potential in specifically killing tumor cells, and offering new possibilities for ovarian cancer (OC) treatment. However, neutralizing antibodies in the ascites from OC patients greatly limit the further application of Reo. In this study, we employed cationic liposomes (Lipo) to deliver Reo, significantly enhancing its ability to enter OC cells and its effectiveness in killing these cells under ascitic conditions. Pre-treatment with the MßCD inhibitor notably decreased Reo-mediated tumor cell death, indicating that Lipo primarily enables Reo's cellular uptake through caveolin-mediated endocytosis. Our results demonstrate that Lipo effectively facilitates the entry of Reo into the cytoplasm and triggers cell apoptosis. The above findings provide a new strategy to overcome the obstacle of neutralizing antibodies in the clinical application of Reo.

Association between twin status with cognitive, behavioral development and brain structure in early adolescence: a retrospective cohort analysis based on the Adolescent Brain Cognitive Development Study.

Twin births are related with maternal and fetal adverse outcomes. Little was known about the comparability of the cognitive, behavioral development and brain structure between twins and singletons in early adolescence. This retrospective cohort study was based on data from the United States population-based, prospective, longitudinal observational Adolescent Brain Cognitive Development study. Children with complete twin status information were enrolled, and the exposure variable was twin status. Primary outcomes were cognitive, behavioral development and brain structure in early adolescence. Cognitive and behavioral outcomes were assessed by using the NIH Toolbox and Child Behavioral Checklist, respectively. Brain structure was evaluated by the cortical thickness, area, and volume extracted from the magnetic resonance imaging (MRI) data. Subgroup analyses were conducted by prematurity, birth weight, with sibling, genetic profiles, and twin types (zygosity). From 1st September 2016 to 15th November 2018, 11545 children (9477 singletons and 2068 twins) aged 9-10 years were enrolled. Twins showed mildly lower cognitive performance (|t|> 5.104, P-values < 0.001, False Discovery Rate [FDR] < 0.001), better behavioral outcome (|t|> 2.441, P-values < 0.015, FDR < 0.042), such as lower scores for multiple psychiatric disorders and behavioral issues, and smaller cortical volume (t = - 3.854, P-values < 0.001, FDR < 0.001) and cortical area (t = - 3.872, P-values < 0.001, FDR < 0.001). The observed differences still held when stratified for prematurity, birth weight, presence of siblings, genetic profiles, and twin types (zygosity). Furthermore, analyses on the two-year follow-up data showed consistent results with baseline data. Twin status is associated with lower cognitive and better behavioral development in early adolescence accompanied by altered brain structure. Clinicians should be aware of the possible difference when generalizing results from adolescent twin samples to singletons.

Advances in clinical research on ultrasound-guided radiofrequency ablation for papillary thyroid microcarcinoma.

Ultrasound-guided radiofrequency ablation (RFA) emerges as a minimally invasive strategy for papillary thyroid microcarcinoma (PTMC), offering advantages over traditional surgical approaches. RFA employs high-frequency electric currents under precise ultrasound guidance to ablate cancerous tissue. Clinical trials consistently demonstrate RFA's efficacy in tumor control and patient-reported outcomes. However, long-term studies are essential to validate its durability and monitor for potential complications. Collaborative efforts among various medical disciplines ensure procedural accuracy and comprehensive postoperative care. Technological innovations, such as enhanced ultrasound imaging and temperature control, promise to refine RFA's precision and effectiveness. Nevertheless, challenges persist, including the need for standardized protocols and comparative studies with traditional treatments. Future research should focus on long-term outcomes, patient selection criteria, and optimization of procedural techniques to solidify RFA's role in PTMC management. RFA presents a promising avenue for PTMC treatment, warranting further investigation and refinement in clinical practice.

Longer-term Effect of Physical Activity Intervention on Sleep Quality: A Secondary Analysis of Cluster Randomized Controlled Trial.

STUDY OBJECTIVES: To examine the longer-term effect of physical activity (PA) intervention on sleep quality and whether the effect was heterogeneous between daytime nappers and non-nappers. METHODS: This study was a secondary analysis of a cluster randomized controlled trial in China. Eight villages were randomized 1:1 to intervention or control group. The intervention group received an 8-week PA intervention, while the control group did not. The primary outcome of this study was the change in the Pittsburgh Sleep Quality Index (PSQI) global score at 24 months. RESULTS: The 511 participants had a mean age of 70.94 years (SD 5.73) and 55.6% were female. The intervention showed improvements in the PSQI global score at 8 weeks (adjusted mean difference -1.05; P=0.002), and the effect diminished at 24 months (-0.64; P=0.06). There were statistically significant improvements in the PSQI global score for daytime nappers, but not for non-nappers at 8 weeks (adjusted mean difference -0.98; P=0.01 vs -1.27; P=0.05), 12 months (-0.86; P=0.03 vs -0.84; P=0.21), and 24 months (-0.80; P=0.04 vs -0.14; P=0.84), although these improvements were below the minimum detectible level of the PSQI which is 1 point. CONCLUSION: The 8-week PA intervention was effective in improving sleep quality, while the effect was diminished and below the minimum detectible level of the PSQI which is 1 point after 24 months. The effect of PA intervention on sleep quality was more pronounced in daytime nappers. Additional interventions (e.g., focusing on multiple behavioral interventions such as PA and healthy diet) are needed to maintain the beneficial effect of PA on sleep quality in the general older populations. Further research is required to confirm the mechanisms of the effect of napping and develop tailored interventions.

[Distribution Characteristics and Risk Assessment of Organophosphates in Water and Sediment in Dongting Lake].

Organophosphates （OPEs） are widely used as flame retardants and additives and thus are commonly detected in the environment. In order to explore their environmental behavior, the concentrations of 13 OPEs in the surface water and sediment of Dongting Lake were analyzed using UPLC-MS/MS. The results showed that 11 OPEs were detected, with detection frequencies of 5.26%-100% and 58.3%-100%, and the concentrations of OPEs were 2.06-2 028 ng·L-1 and 19.6-2 232 ng·g-1 in water and sediment, respectively. Overall, contamination concentrations were ranked in descending order as follows： inflowing rivers, lake area, and outlet, whereas the spatial distribution of concentrations in sediment was inversely proportional to hydrodynamics. The concentration of OPEs in Dongting Lake was at a high level compared with that of domestic and foreign lakes. Among the detected 11 OPEs, tri-iso-butyl phosphate （TnBP） and （TiBP） were dominant in water, accounting for 52.3% and 22.4% of ∑OPEs, respectively. TPhP was the dominant OPEs in sediment, accounting for 31.2% of ∑OPEs. The correlation and principal component analysis indicated that OPEs pollution in Dongting Lake was mainly affected by industrial production emissions, fishery aquaculture, and atmospheric deposition. The assessment results of the risk entropy showed that most of the detected OPEs in water had relatively low ecological risks, whereas the ecological risk of 2-ethylhexyl diphenyl phosphate （EHDPP） at some sampling points requires further attention.

Lifestyle factors and their relative contributions to longitudinal progression of cardio-renal-metabolic multimorbidity: a prospective cohort study.

BACKGROUND: The role of lifestyle factors and their relative contributions to the development and mortality of cardio-renal-metabolic multimorbidity (CRMM) remains unclear. METHODS: A study was conducted with 357,554 UK Biobank participants. CRMM was defined as the coexistence of two or three cardio-renal-metabolic diseases (CRMDs), including cardiovascular disease (CVD), type 2 diabetes (T2D) and chronic kidney disease (CKD). The prospective study examined the associations of individual and combined lifestyle scores (diet, alcohol consumption, smoking, physical activity, sedentary behavior, sleep duration and social connection) with longitudinal progression from healthy to first cardio-renal-metabolic disease (FCRMD), then to CRMM, and ultimately to death, using a multistate model. Subsequently, quantile G-computation was employed to assess the relative contribution of each lifestyle factor. RESULTS: During a median follow-up of 13.62 years, lifestyle played crucial role in all transitions from healthy to FCRMD, then to CRMM, and ultimately to death. The hazard ratios (95% CIs) per score increase were 0.91 (0.90, 0.91) and 0.90 (0.89, 0.91) for healthy to FCRMD, and for FCRMD to CRMM, and 0.84 (0.83, 0.86), 0.87 (0.86, 0.89), and 0.90 (0.88, 0.93) for mortality risk from healthy, FCRMD, and CRMM, respectively. Among the seven factors, smoking status contributed to high proportions for the whole disease progression, accounting for 19.88-38.10%. High-risk diet contributed the largest proportion to the risk of transition from FCRMD to CRMM, with 22.53%. Less-frequent social connection contributed the largest proportion to the risk of transition from FCRMD to death, with 28.81%. When we further consider the disease-specific transitions, we find that lifestyle scores had slightly stronger associations with development to T2D than to CVD or CKD. CONCLUSIONS: Our study indicates that a healthy lifestyle may have a protective effect throughout the longitudinal progression of CRMM, informing more effective management and treatment. Smoking status, diet, and social connection played pivotal roles in specific disease transitions.

Comprehensive Analysis of Immune Cell Infiltration and M2-Like Macrophage Biomarker Expression Patterns in Atrial Fibrillation.

Background: Macrophages play a crucial role in the progression of AF, closely linked to atrial inflammation and myocardial fibrosis. However, the functions and molecular mechanisms of different phenotypic macrophages in AF are not well understood. This study aims to analyze the infiltration characteristics of atrial immune cells in AF patients and further explore the role and molecular expression patterns of M2 macrophage-related genes in AF. Methods: This study integrates single-cell and large-scale sequencing data to analyze immune cell infiltration and molecular characterization of the LAA in patients with AF, using SR as a control group. CIBERSORT assesses immune cell types in LAA tissues; WGCNA identifies signature genes; cell clustering analyzes cell types and subpopulations; cell communication explores macrophage interactions; hdWGCNA identifies M2 macrophage gene modules in AF. AF biomarkers are identified using LASSO and Random Forest, validated with ROC curves and RT-qPCR. Potential molecular mechanisms are inferred through TF-miRNA-mRNA networks and single-gene enrichment analyses. Results: Myeloid cell subsets varied considerably between the AF and SR groups, with a significant increase in M2 macrophages in the AF group. Signals of inflammation and matrix remodeling were observed in AF. M2 macrophage-related genes IGF1, PDK4, RAB13, and TMEM176B were identified as AF biomarkers, with RAB13 and TMEM176B being novel markers. A TF-miRNA-mRNA network was constructed using target genes, which are enriched in the PPAR signaling pathway and fatty acid metabolism. Conclusion: Over infiltration of M2 macrophages may be an important factor in the progression of AF. The M2 macrophage-related genes IGF1, RAB13, TMEM176B and PDK4 may regulate the progression of AF through the PPAR signaling pathway and fatty acid metabolism.

Mechanism Switch in Surface-Enhanced Raman Scattering: The Role of Nanoparticle Dimensions.

Surface-enhanced Raman scattering (SERS) is renowned for amplifying Raman signals, with electromagnetic mechanism (EM) enhancement arising from localized surface plasmon resonances and chemical mechanism (CM) enhancement as a result of charge transfer interactions. Despite the conventional emphasis on EM as a result of plasmonic effects, recent findings highlight the significance of CM when noble metals appear as smaller entities. However, the threshold size of the noble metal clusters/particles corresponding to the switch in SERS mechanisms is not clear at present. In this work, the VSe2-xOx/Au composites with different Au sizes are employed, in which a clear view of the SERS mechanism switch is observed at the Au size range of 16-21 nm. Our findings not only provide insight into the impact of noble metal size on SERS efficiency but also offer quantitative data to assist researchers in making informed judgments when analyzing SERS mechanisms.

VirRep: a hybrid language representation learning framework for identifying viruses from human gut metagenomes.

Identifying viruses from metagenomes is a common step to explore the virus composition in the human gut. Here, we introduce VirRep, a hybrid language representation learning framework, for identifying viruses from human gut metagenomes. VirRep combines a context-aware encoder and an evolution-aware encoder to improve sequence representation by incorporating k-mer patterns and sequence homologies. Benchmarking on both simulated and real datasets with varying viral proportions demonstrates that VirRep outperforms state-of-the-art methods. When applied to fecal metagenomes from a colorectal cancer cohort, VirRep identifies 39 high-quality viral species associated with the disease, many of which cannot be detected by existing methods.

Population coding for figure-ground texture segregation in macaque V1 and V4.

Object recognition often involves the brain segregating objects from their surroundings. Neurophysiological studies of figure-ground texture segregation have yielded inconsistent results, particularly on whether V1 neurons can perform figure-ground texture segregation or just detect texture borders. To address this issue from a population perspective, we utilized two-photon calcium imaging to simultaneously record the responses of large samples of V1 and V4 neurons to figure-ground texture stimuli in awake, fixating macaques. The average response changes indicate that V1 neurons mainly detect texture borders, while V4 neurons are involved in figure-ground segregation. However, population analysis (SVM decoding of PCA-transformed neuronal responses) reveal that V1 neurons not only detect figure-ground borders, but also contribute to figure-ground texture segregation, although requiring substantially more principal components than V4 neurons to reach a 75 % decoding accuracy. Individually, V1/V4 neurons showing larger (negative/positive) figure-ground response differences contribute more to figure-ground segregation. But for V1 neurons, the contribution becomes significant only when many principal components are considered. We conclude that V1 neurons participate in figure-ground segregation primarily by defining the figure borders, and the poorly structured figure-ground information V1 neurons carry could be further utilized by V4 neurons to accomplish figure-ground segregation.

In Situ Preparation of Metallic Copper Nanosheets/Carbon Paper Sensitive Electrodes for Low-Potential Electrochemical Detection of Nitrite.

In the realm of electrochemical nitrite detection, the potent oxidizing nature of nitrite typically necessitates operation at high detection potentials. However, this study introduces a novel approach to address this challenge by developing a highly sensitive electrochemical sensor with a low reduction detection potential. Specifically, a copper metal nanosheet/carbon paper sensitive electrode (Cu/CP) was fabricated using a one-step electrodeposition method, leveraging the catalytic reduction properties of copper's high occupancy d-orbital. The Cu/CP sensor exhibited remarkable performance in nitrite detection, featuring a low detection potential of -0.05 V vs. Hg/HgO, a wide linear range of 10~1000 µM, an impressive detection limit of 0.079 µM (S/N = 3), and a high sensitivity of 2140 µA mM-1cm-2. These findings underscore the efficacy of electrochemical nitrite detection through catalytic reduction as a means to reduce the operational voltage of the sensor. By showcasing the successful implementation of this strategy, this work sets a valuable precedent for the advancement of electrochemical low-potential nitrite detection methodologies.

Plasmonic trimers designed as SERS-active chemical traps for subtyping of lung tumors.

Plasmonic materials can generate strong electromagnetic fields to boost the Raman scattering of surrounding molecules, known as surface-enhanced Raman scattering. However, these electromagnetic fields are heterogeneous, with only molecules located at the 'hotspots', which account for ≈ 1% of the surface area, experiencing efficient enhancement. Herein, we propose patterned plasmonic trimers, consisting of a pair of plasmonic dimers at the bilateral sides and a trap particle positioned in between, to address this challenge. The trimer configuration selectively directs probe molecules to the central traps where 'hotspots' are located through chemical affinity, ensuring a precise spatial overlap between the probes and the location of maximum field enhancement. We investigate the Raman enhancement of the Au@Al2O3-Au-Au@Al2O3 trimers, achieving a detection limit of 10-14 M of 4-methylbenzenethiol, 4-mercaptopyridine, and 4-aminothiophenol. Moreover, single-molecule SERS sensitivity is demonstrated by a bi-analyte method. Benefiting from this sensitivity, our approach is employed for the early detection of lung tumors using fresh tissues. Our findings suggest that this approach is sensitive to adenocarcinoma but not to squamous carcinoma or benign cases, offering insights into the differentiation between lung tumor subtypes.