ZBP1 condensate formation synergizes Z-NAs recognition and signal transduction.

Z-DNA binding protein 1 (ZBP1) is a crucial player in the intracellular recognition of Z-form nucleic acids (Z-NAs) through its Zαß domain, initiating downstream interactions with RIPK1 and RIPK3 via RHIM domains. This engagement leads to the assembly of PANoptosomes, ultimately inducing programmed cell death to curb pathogen dissemination. How Zαß and RHIM domain cooperate to trigger Z-NAs recognition and signal transduction remains unclear. Here, we show that ZBP1 condensate formation facilitates Z-NAs binding and antiviral signal transduction. The ZBP1 Zαß dimerizes in a concentration-dependent manner, forming characteristic condensates in solutions evidenced by DLS and SAXS methods. ZBP1 exhibits a binding preference for 10-bp length CG (10CG) DNA and Z-RNA ligand, which in turn enhanced Zαß dimerization, expediting the formation of droplet condensates in vitro and amyloid-like puncta in cells. Subsequent investigations reveal that Zαß could form condensates with liquid-liquid phase separation property upon HSV and IAV infections, while full-length ZBP1 forms amyloid-like puncta with or without infections. Furthermore, ZBP1 RHIM domains show typical amyloidal fibril characterizations and cross-polymerize with RIPK1 depending on the core motif of 206IQIG209, while mutated ZBP1 could impede necroptosis and antiviral immunity in HT-29 cells. Thus, ZBP1 condensate formation facilitates the recognition of viral Z-NAs and activation of downstream signal transduction via synergic action of different domains, revealing its elaborated mechanism in innate immunity.

Two-stage fermentation of corn and soybean meal mixture by Bacillus subtilis and Lactobacillus acidophilus to improve feeding value: optimization, physicochemical property, and microbial community.

Microbial treatment can reduce the antinutritional factors and allergenic proteins in corn-soybean meal mixture (CSMM), but the role of the microbial community in hypoallergenicity and digestibility during the fermentation process remains unclear. Therefore, the fermentation strains of Bacillus and LAB were determined, and the compatibility and fermentation process of two-stage solid fermentation composite bacteria were optimized, and the dynamic changes in physicochemical property and microbial community during two-stage fermentation were investigated. Results showed that Bacillus subtilis NCUBSL003 and Lactobacillus acidophilus NCUA065016 were the best fermentation combinations. The optimal fermentation conditions were inoculum 7.14%, solid-liquid ratio of 1:0.88 and fermentation time of 74.30 h. The contents of TI, ß-conglycinin and glycinin decreased significantly after fermentation. Besides, TCA-SP, small peptides and FAA increased. Bacillus and Lactobacillus were the main genera. Pathogenic bacteria genera were inhibited effectively. This study suggests the feasibility of two-stage fermentation in improving the nutrient values and safety of the CSMM. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-023-01426-7.

Two-photon fluorescence-guided precise photothermal therapy located in a single cancer cell utilizing bifunctional N-doped carbon quantum dots.

The utilization of carbon quantum dots (CQDs) for photothermal therapy has emerged as a hot research topic. However, there has been limited research on killing one single cancer cell which is critical in reducing unnecessary damage to the surrounding healthy tissues. In this work, we developed a two-photon fluorescence-guided precise photothermal therapy in a single human malignant melanoma (A375) cancer cell utilizing bifunctional N-doped CQDs. Resulting from the two-photon fluorescence of the CQDs, one single cancer cell can be located and simultaneously destroyed by the photothermal effect of the same CQDs. Specifically, the balanced two-photon absorption cross-section (7000 GM) and photoluminescence quantum yield (8.4%) of the CQDs enable the fluorescence-guided photothermal treatment to be achieved in only 5 s under the irradiation of 800 nm laser of 27.5 mW, much faster than the control experiment without the guidance of fluorescence. The heat generated by the aggregated CQDs is in sufficient amounts while being confined in a small area, as evidenced by the numerical simulations and photothermal experiments, to limit the range of thermal treatment in the cells. This work provides a new approach for realizing photothermal therapy with minimal damage and establishes a new application scenario of CQDs for precise tumor ablation.

Effects of fermentation with Lactiplantibacillus plantarum NCU116 on the antihypertensive activity and protein structure of black sesame seed.

Effects of fermentation by Lactobacillus Plantarum NCU116 on the antihypertensive potential of black sesame seed (BSS) and structure characteristics of fermented black sesame seed protein (FBSSP) were investigated. Angiotensin-I-converting enzyme (ACE) inhibition and zinc chelating ability of fermented black sesame seed hydrolysate (FBSSH) reached the highest of 60.78 ± 3.67 % and 2.93 ± 0.04 mg/mL at 48 h and 60 h of fermentation, respectively. Additionally, the antioxidant activities of FBSSH and surface hydrophobicity of FBSSP were increased noticeably by fermentation. The α-helix and ß-rotation of FBSSP tended to decrease and increase, respectively, during fermentation. Correlation analysis indicated strong positive relationships between ß-turn and ACE inhibition activity as well as zinc chelating ability with correlation coefficients r of 0.8976 and 0.8932. Importantly, novel ACE inhibitory peptides LLLPYY (IC50 = 12.20 µM) and ALIPSF (IC50 = 558.99 µM) were screened from FBSSH at 48 h using in silico method. Both peptides showed high antioxidant activities in vitro. Molecular docking analysis demonstrated that the hydrogen bond connected with zinc ions of ACE mainly attributed to the potent ACE inhibitory activity of LLLPYY. The findings indicated that fermentation by Lactobacillus Plantarum NCU116 is an effective method to enhance the antihypertensive potential of BSS.

Brain region changes following a spinal cord injury.

Brain-related complications are common in clinical practice after spinal cord injury (SCI); however, the molecular mechanisms of these complications are still unclear. Here, we reviewed the changes in the brain regions caused by SCI from three perspectives: imaging, molecular analysis, and electrophysiology. Imaging studies revealed abnormal functional connectivity, gray matter volume atrophy, and metabolic abnormalities in brain regions after SCI, leading to changes in the structure and function of brain regions. At the molecular level, chemokines, inflammatory factors, and damage-associated molecular patterns produced in the injured area were retrogradely transmitted through the corticospinal tract, cerebrospinal fluid, or blood circulation to the specific brain area to cause pathologic changes. Electrophysiologic recordings also suggested abnormal changes in brain electrical activity after SCI. Transcranial magnetic stimulation, transcranial direct current stimulation, and deep brain stimulation alleviated pain and improved motor function in patients with SCI; therefore, transcranial therapy may be a new strategy for the treatment of patients with SCI.

Integrated metatranscriptomics and metabolomics reveal microbial succession and flavor formation mechanisms during the spontaneous fermentation of Laotan Suancai.

Laotan Suancai, a Chinese traditional fermented vegetable, possesses a unique flavor that depends on the fermentative microbiota. However, the drivers of microbial succession and the correlation between flavor and active microbiota remain unclear. A total of 21 characteristic flavor metabolites were identified in Laotan Suancai by metabolomics, including 8 sulfides, 6 terpenes, 3 organic acids, 2 isothiocyanates, 1 ester, and 1 pyrazine. Metatranscriptome analysis revealed variations in the active microbiota at different stages of fermentation, and further analysis indicated that organic acids were the primary drivers of microbial succession. Additionally, we reconstructed the metabolic network responsible for the formation of characteristic flavor compounds and identified Companilactobacillus alimentarius, Weissella cibaria, Lactiplantibacillus plantarum, and Loigolactobacillus coryniformis as the core functional microbes involved in flavor development. This study contributed to profoundly understanding the relationship between the active microbiota and flavor quality formation, as well as the targeted selection of starters with flavor regulation abilities.

Dynamic conformational changes of a tardigrade group-3 late embryogenesis abundant protein modulate membrane biophysical properties.

A number of intrinsically disordered proteins (IDPs) encoded in stress-tolerant organisms, such as tardigrade, can confer fitness advantage and abiotic stress tolerance when heterologously expressed. Tardigrade-specific disordered proteins including the cytosolic-abundant heat-soluble proteins are proposed to confer stress tolerance through vitrification or gelation, whereas evolutionarily conserved IDPs in tardigrades may contribute to stress tolerance through other biophysical mechanisms. In this study, we characterized the mechanism of action of an evolutionarily conserved, tardigrade IDP, HeLEA1, which belongs to the group-3 late embryogenesis abundant (LEA) protein family. HeLEA1 homologs are found across different kingdoms of life. HeLEA1 is intrinsically disordered in solution but shows a propensity for helical structure across its entire sequence. HeLEA1 interacts with negatively charged membranes via dynamic disorder-to-helical transition, mainly driven by electrostatic interactions. Membrane interaction of HeLEA1 is shown to ameliorate excess surface tension and lipid packing defects. HeLEA1 localizes to the mitochondrial matrix when expressed in yeast and interacts with model membranes mimicking inner mitochondrial membrane. Yeast expressing HeLEA1 shows enhanced tolerance to hyperosmotic stress under nonfermentative growth and increased mitochondrial membrane potential. Evolutionary analysis suggests that although HeLEA1 homologs have diverged their sequences to localize to different subcellular organelles, all homologs maintain a weak hydrophobic moment that is characteristic of weak and reversible membrane interaction. We suggest that such dynamic and weak protein-membrane interaction buffering alterations in lipid packing could be a conserved strategy for regulating membrane properties and represent a general biophysical solution for stress tolerance across the domains of life.

Efficient optical plasmonic tweezer-controlled single-molecule SERS characterization of pH-dependent amylin species in aqueous milieus.

It is challenging to characterize single or a few biomolecules in physiological milieus without excluding the influences of surrounding environment. Here we utilize optical plasmonic trapping to construct a dynamic nanocavity, which reduces the diffraction-limited detection volume and provides reproducible electromagnetic field enhancements to achieve high-throughput single-molecule surface-enhanced Raman spectroscopy (SERS) characterizations in aqueous environments. Specifically, we study human Islet Amyloid Polypeptide (amylin, hIAPP) under different physiological pH conditions by combining spectroscopic experiments and molecular dynamics (MD) simulations. Based on a statistically significant amount of time-dependent SERS spectra, two types of low-populated transient species of hIAPP containing either turn or ß-sheet structure among its predominant helix-coil monomers are characterized during the early-stage incubation at neutral condition, which play a crucial role in driving irreversible amyloid fibril developments even after a subsequent adjustment of pH to continue the prolonged incubation at acidic condition. Our results might provide profound mechanistic insight into the pH-regulated amyloidogenesis and introduce an alternative approach for investigating complex biological processes at the single-molecule level.

Effects of enzyme treatment on the antihypertensive activity and protein structure of black sesame seed (Sesamum indicum L.) after fermentation pretreatment.

Effects of enzyme treatment on the hypertensive potential and protein structure of black sesame seed (BSS) were investigated. Compared with BSS, Angiotensin-converting enzyme (ACE) inhibition of fermented black sesame seed (FBSS) has significantly improved after acid protease processing and reached 75.39% at 2 U/g in 3 h. Meanwhile, the zinc chelating ability and antioxidant activity of FBSS hydrolysate as well as surface hydrophobicity, free sulfhydryl content, and peptide content of FBSS protein, were significantly increased. The results illustrated that this strategy promoted the protein unfolding and exposure of hydrophobic residues, thus contributing toward enzymatic hydrolysis. Secondary structure results indicated that the α-helix of FBSS protein and ß-sheet of BSS protein decreased after hydrolyzing. The differences in ACE inhibition may also result from the difference in peptide sequence except for peptide content. In conclusion, the combination of fermentation pretreatment and enzyme treatment is an effective method to enhance the antihypertensive potential of BSS.

Dynamics of bacterial community, metabolites profile and physicochemical characteristics during solid-state fermentation of soybean meal and corn mixed substrates inoculated with Bacillus pumilus and Limosilactobacillus fermentum.

BACKGROUND: Solid-state fermentation (SSF) is a general approach for preparing food and feed, which not only improves nutrition but also provides prebiotics and metabolites. Although many studies have been conducted on the effects of fermentation on feed substrate, the dynamics of microbiota and metabolites in SSF remain unclear. Here, high-throughput sequencing combined with gas chromatography-quadrupole time-of-flight mass spectrometry was used to evaluate the dynamic changes of solid fermented soybean meal and corn mixed matrix inoculated with Bacillus pumilus and Limosilactobacillus fermentum. RESULTS: Generally, inoculated bacteria rapidly proliferated, accompanied by the degradation of macromolecular proteins and an increase in the content of small peptides, trichloroacetic acid-soluble protein, free amino acids and organic acids. Bacillus, Lactobacillus and Enterococcus dominated the whole fermentation process. 389 non-volatile metabolites and 182 volatile metabolites were identified, including amino acids, organic acids, ketones, aldehydes, furans and pyrazine. Typical non-volatile metabolites such as lactic acid, 4-aminobutanoic acid, l-glutamic acid, d-arabinose and volatile metabolites such as 4-ethyl-2-methoxyphenol, 4-penten-2-ol, 2-pentanone, 2-ethylfuran, 2-methylhexanoic acid and butanoic acid-ethyl ester were significantly increased in two-stage solid fermentation. However, some adverse metabolites were also produced, such as oxalic acid, acetic acid, tyramine and n-butylamine, which may affect the quality of fermented feed. Sixteen genera were significantly correlated with differential non-volatile metabolites, while 11 genera were significantly correlated with differential volatile metabolites. CONCLUSION: These results characterized the dynamic changes in the process of two-stage solid-state fermentation with Bacillus pumilus and Limosilactobacillus fermentum and provided a potential reference for additional intervention on improving the effectiveness and efficiency of solid-state fermentation of feed in the future. © 2023 Society of Chemical Industry.

Tunable lipid-coated nanoporous silver sheet for characterization of protein-membrane interactions by surface-enhanced Raman scattering (SERS).

Membrane environments affect protein structures and functions through protein-membrane interactions in a wide range of important biological processes. To better study the effects from the lipid's hydrophilic and hydrophobic interaction with protein on different membrane regions, we developed the lipid-coated nanoporous silver sheets to provide tunable supported lipid monolayer/bilayer environments for in situ surface-enhanced Raman vibrational spectroscopy (SERS) characterizations. Under the controllable surface pressure, lipid monolayer/bilayer was coated along the microscopic curved surface of nanoporous silver sheets to serve as a cell membrane mimic as well as a barrier to avoid protein denaturation while empowering the high SERS enhancements from the underlying metallic bases allowing detection sensitivity at low physiological concentrations. Moreover, we fine-tuned the lipid packing density and controlled the orientation of the deposited lipid bilayers and monolayers to directly monitor the protein structures upon interactions with various membrane parts/positions. Our results indicate that lysozyme adopted the α-helical structure in both hydrophilic and hydrophobic interaction with lipid membrane. Interestingly, alpha-synuclein folded into the α-helical structure on the negatively charged lipid heads, whereas the hydrophobic lipid tails induced the ß-sheet structural conversion of alpha-synuclein originated from its unstructured monomers. These direct observations on protein hydrophilic and hydrophobic interaction with lipid membrane might provide profound insights into the formation of the ß-sheet-containing alpha-synuclein oligomers for further membrane disruptions and amyloid genesis associated with Parkinson's disease. Hence, with the controllability and tunability of lipid environments, our platform holds great promise for more general applications in investigating the influences from membranes and the correlative structures of proteins under both hydrophilic and hydrophobic effects.

Construction of multiphasic membraneless organelles towards spontaneous spatial segregation and directional flow of biochemical reactions.

Many intracellular membraneless organelles (MLOs) appear to adapt a hierarchical multicompartment organization for efficient coordination of highly complex reaction networks. Recapitulating such an internal architecture in biomimetic platforms is, therefore, an important step to facilitate the functional understanding of MLOs and to enable the design of advanced microreactors. Herein, we present a modular bottom-up approach for building synthetic multiphasic condensates using a set of engineered multivalent polymer-oligopeptide hybrids. These hybrid constructs exhibit dynamic phase separation behaviour generating membraneless droplets with a subdivided interior featuring distinct chemical and physical properties, whereby a range of functional biomolecules can be spontaneously enriched and spatially segregated. The platform also attains separated confinement of transcription and translation reactions in proximal compartments, while allowing inter-compartment communication via a directional flow of reactants. With advanced structural and functional features attained, this system can be of great value as a MLO model and as a cell-free system for multiplex chemical biosynthesis.

Correction: Highly reusable nanoporous silver sheet for sensitive SERS detection of pesticides.

Correction for 'Highly reusable nanoporous silver sheet for sensitive SERS detection of pesticides' by Huanyu Chi et al., Analyst, 2020, 145, 5158-5165, https://doi.org/10.1039/D0AN00999G.

Comparison of nutritional value, bioactivity, and volatile compounds of soybean meal-corn bran mixed substrates fermented by different microorganisms.

To evaluate the impact of fermentation with different microorganisms on the nutritional quality and bioactivity of soybean meal-corn bran mixed substrates (MS), five lactic acid bacteria (LAB) strains, two Bacillus, and two yeast strains with excellent probiotics were selected for solid-state fermentation of soybean meal and corn bran MS. The fermented mixed substrate (FMS) inoculated with Lacticaseibacillus casei, Lactobacillus fermentum, Lactiplantibacillus plantarum, and Lactobacillus acidophilus presents lower risk of infection with pathogenic bacteria, probably due to their low pH and high lactate content. Compared to the FMS with LAB and yeast, Bacillus subtilis and B. pumilus showed significant improvements in nutritional quality and bioactivity, including TCA-SP, small peptide, free amino acids, total phenol, and protein digestibility. More than 300 volatile compounds were identified in FMS, including alcohols, ketones, aldehydes, esters, acids, ethers, furans, pyrazines, benzene, phenols, amines, alkanes, and others. FMS with Bacillus was characterized as containing a greater number of compounds such as ketones, aldehydes, and pyrazines. This study showed that microbial fermented feeds differed with various microorganism, and fermentation was an effective way to improve the quality of soybean meal-corn bran mixed feeds. This study might be the basis for excellent strains screening for multi-microbial combined fermentation in the future.

Moderate Binding between Two SARS-CoV-2 Protein Segments and α-Synuclein Alters Its Toxic Oligomerization Propensity Differently.

The neurological symptoms of long COVID and viral neuroinvasion have raised concerns about the potential interactions between SARS-CoV-2 protein segments and neuronal proteins, which might confer a risk of post-infection neurodegeneration, but the underlying mechanisms remain unclear. Here, we reported that the receptor-binding domain (RBD) of the spike protein and the nine-residue segment (SK9) of the envelope protein could bind to α-synuclein (αSyn) with Kd values of 503 ± 24 nM and 12.7 ± 1.6 µM, respectively. RBD could inhibit αSyn fibrillization by blocking the non-amyloid-ß component region and mediating its antiparallel ß-sheet structural conversions. Omicron-RBD (BA.5) was shown to have a slightly stronger affinity for αSyn (Kd = 235 ± 10 nM), which implies similar effects, whereas SK9 may bind to the C-terminus which accelerates the formation of parallel ß-sheet-containing oligomers and abruptly increases the rate of membrane disruption by 213%. Our results provide plausible molecular insights into the impact of SARS-CoV-2 post-infection and the oligomerization propensity of αSyn that is associated with Parkinson's disease.

Directed assembly of genetically engineered eukaryotic cells into living functional materials via ultrahigh-affinity protein interactions.

Engineered living materials (ELMs) are gaining traction among synthetic biologists, as their emergent properties and nonequilibrium thermodynamics make them markedly different from traditional materials. However, the aspiration to directly use living cells as building blocks to create higher-order structures or materials, with no need for chemical modification, remains elusive to synthetic biologists. Here, we report a strategy that enables the assembly of engineered Saccharomyces cerevisiae into self-propagating ELMs via ultrahigh-affinity protein/protein interactions. These yeast cells have been genetically engineered to display the protein pairs SpyTag/SpyCatcher or CL7/Im7 on their surfaces, which enable their assembly into multicellular structures capable of further growth and proliferation. The assembly process can be controlled precisely via optical tweezers or microfluidics. Moreover, incorporation of functional motifs such as super uranyl-binding protein and mussel foot proteins via genetic programming rendered these materials suitable for uranium extraction from seawater and bioadhesion, respectively, pointing to their potential in chemical separation and biomedical applications.

The Focus and New Progress of Percutaneous Balloon Compression for the Treatment of Trigeminal Neuralgia.

Trigeminal neuralgia is a condition confined to the trigeminal nerve, causing one or more branches of facial nerve pain. Surgical treatment options for trigeminal neuralgia include microvascular decompression(MVD), percutaneous balloon compression (PBC), radiofrequency thermocoagulation(RF), percutaneous retrogasserian glycerol rhizotomy(PRGR), gamma knife, etc. Of these treatments, PBC is increasingly being used by clinicians for trigeminal neuralgia. PBC is a simple surgical operation performed to treat trigeminal neuralgia. Owing to its advantages, PBC is favored by many clinicians. In this study, we aimed to emphasize the need to analyze the shape of the balloon, position, compression time, and pressure, as these factors can affect the efficacy of PBC. The relief of pain by balloon compression is related to the shape of the balloon on X-ray, which is the key to the operation. Owing to continued progress and advances in current imaging technologies, clinicians revealed that the precise positioning of the foramen ovale is no longer an intraoperative problem. Instead, the anatomy of Meckel's cave and the shape of the balloon must be the focus to achieve the best treatment effect. For clinicians, PBC is simple and is associated with a short operation time. PBC also has other advantages, such as low cost and immediate postoperative pain relief. The recurrence rate of pain post-PBC is low, despite the occurrence of facial numbness post-op. However, this side effect is reversible and does not affect daily life of the patient. In fact, the patient can be discharged 1-2 days after surgery. Overall, PBC can be considered as one of the preferred surgical methods for the treatment of primary trigeminal neuralgia. In this paper, we explain the main points of PBC operation in detail in terms of Meckel's cave, surgical procedure, complications, discussion of the focus and new progress, etc.

FOXO1 represses MCL1 transcription to regulate the function of vascular smooth muscle cells in intracranial aneurysm.

Intracranial aneurysm (IA) is a pathological dilation of the cerebral arteries. Vascular smooth muscle cell (VSMC) dysfunction assumes a role in IA development. In this context, this study probed the role of FOXO1 in human brain VSMC (HBVSMC) function via MCL1. FOXO1 and MCL1 expression in arterial wall tissues from IA patients and inflammatory cytokines (IL-1ß, TNF-α, and IL-6) levels in the serum of IA patients were, respectively, detected with qRT-PCR and ELISA. Pearson's correlation analysis was utilized to analyze the correlation between FOXO1 and MCL1. After FOXO1 and/or MCL1 were overexpressed in HBVSMCs, caspase-3 and Cyt-c protein expression were examined by western blot, cell proliferation by CCK-8 and EdU assays, and cell apoptosis by flow cytometry. IL-1ß, TNF-α, and IL-6 levels were assessed in the supernatant of HBVSMCs with ELISA. Dual-luciferase gene reporter and ChIP assays were conducted to evaluate the binding of FOXO1 to MCL1. FOXO1 expression was high and MCL expression was low in arterial wall tissues from IA patients, and IL-1ß, TNF-α, and IL-6 levels were high in the serum of IA patients. There was an inverse correlation between FOXO1 and MCL1 mRNA levels. Moreover, FOXO1 bound to the MCL1 promoter to decrease MCL1 transcription. In addition, FOXO1 overexpression augmented cell apoptosis, caspase-3 and Cyt-c protein expression, and IL-1ß, TNF-α, and IL-6 secretion, while reducing cell proliferation in HBVSMCs, which was abrogated by further MCL1 overexpression. FOXO1 impeded MCL1 transcription to curb HBVSMC proliferation and facilitate their apoptosis and inflammation.

Protein purification, crystallization, and structure determination of human DEAD-box RNA helicase DDX21 in different unwinding states.

RNA helicase DDX21 plays vital roles in ribosomal RNA processing and the regulation of host innate immunity during virus infection. Here, we describe the optimized protocols for nucleic acid-free protein purification and crystallization of DDX21 in its different unwinding states. Rational design of the flexible region within the helicase core, and biophysical approach to characterize interactions between DDX21 and RNA, leads to successful crystallization of DDX21. This protocol can be applied to the crystallography of other DExD/H-box RNA helicases. For complete details on the use and execution of this protocol, please refer to Chen et al. (2020).

Optical Trapping of Plasmonic Nanoparticles for In Situ Surface-Enhanced Raman Spectroscopy Characterizations.

Surface-enhanced Raman spectroscopy (SERS) enables the ultrasensitive detection of analyte molecules in various applications due to the enhanced electric field of metallic nanostructures. Salt-induced silver nanoparticle aggregation is the most popular method for generating SERS-active substrates; however, it is limited by poor reproducibility, stability, and biocompatibility. The present protocol integrates optical manipulation and SERS detection to develop an efficient analytical platform to address this. A 1064 nm trapping laser and a 532 nm Raman probe laser are combined in a microscope to assemble silver nanoparticles, which generate plasmonic hotspots for in situ SERS measurements in aqueous environments. Without aggregating agents, this dynamic plasmonic silver nanoparticle assembly enables an approximately 50-fold enhancement of the analyte molecule signal. Moreover, it provides spatial and temporal control to form the SERS-active assembly in as low as 0.05 nM analyte-coated silver nanoparticle solution, which minimizes the potential perturbation for in vivo analysis. Hence, this optical trapping-integrated SERS platform holds great potential for efficient, reproducible, and stable molecular analyses in liquids, especially in aqueous physiological environments.