Exosome Particle Profiling & Physicochemical Analysis
OverviewServicesSamplesAdvantagesApplicationsCase StudyFAQs
Overview
At Creative BioMart Microbe, we provide comprehensive particle profiling and physicochemical analysis services purpose-built for microbial extracellular vesicles (mEVs), including bacterial outer membrane vesicles (OMVs), cytoplasmic membrane vesicles (CMVs) from Gram-positive bacteria, fungal EVs, and phage-induced membrane vesicles. Our platform integrates nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and zeta potential measurement into a single, standardized quality analytics workflow that delivers quantitative, reproducible characterization data aligned with industry standards for extracellular vesicle studies.
Unlike generic exosome analytics services that apply mammalian protocols to microbial samples, we have optimized every sample preparation step, instrument parameter, and data interpretation criterion for the unique biophysical properties of mEVs. From culture supernatant to publication-ready report, clients receive validated particle concentration, size distribution, morphology, and surface charge data that supports CMC documentation, lot-release testing, and regulatory submissions. Contact us to discuss your specific sample type and analytical requirements.
Figure 1. Schematic overview of the integrated particle profiling and physicochemical analysis platform for microbial extracellular vesicles, spanning NTA particle size and concentration profiling, TEM morphology and ultrastructure imaging, and zeta potential surface charge analysis.
Services
Service Workflow
Service Details
NTA Particle Size & Concentration Profiling
We employ NanoSight or equivalent nanoparticle tracking analysis systems to quantify mEV particle concentration and size distribution in liquid suspension. Each sample is diluted in particle-free PBS and analyzed under optimized camera level and detection threshold settings calibrated for microbial vesicles (typical diameter range: 20–400 nm). Results include mean/median particle diameter, mode, D10/D50/D90 percentiles, and particle concentration (particles/mL). For OMV samples, we apply optimized refractive index settings calibrated for lipid-rich microbial vesicles to improve concentration accuracy.
TEM Morphology & Ultrastructure Imaging
We perform negative-stain transmission electron microscopy to visualize mEV morphology, membrane integrity, and purity. Samples are deposited on carbon-coated copper grids, stained with 2% uranyl acetate, and imaged at 80–120 kV accelerating voltage. Our TEM service delivers high-resolution micrographs showing characteristic vesicle ultrastructure (spherical, cup-shaped morphology for OMVs; variable shapes for fungal EVs), enabling visual confirmation of vesicle identity and detection of non-vesicular contaminants (cell debris, protein aggregates, flagella fragments).
Zeta Potential & Surface Charge Analysis
We measure electrophoretic mobility using phase analysis light scattering (PALS) to determine zeta potential, a critical indicator of mEV colloidal stability and surface charge properties. Measurements are performed in low-conductivity buffers at controlled temperature (25°C) with automatic voltage selection. Zeta potential values inform formulation development: highly negative values (–30 to –50 mV) indicate stable OMV suspensions, while shifts toward neutral suggest aggregation risk or surface modification success.
Integrated Data Reporting & Statistical Analysis
All three analytical modalities are consolidated into a unified report with cross-validated metrics. NTA concentration data is correlated with TEM particle counts per field of view; zeta potential trends are mapped against size distribution stability indices. Statistical analysis includes coefficient of variation (CV) across technical replicates, confidence intervals for mean diameter, and outlier detection algorithms. Reports are delivered in PDF and raw data formats (CSV, AVI for NTA, TIFF for TEM).
Batch-to-Batch Consistency & QC Trending
For clients with recurring production batches, we establish QC trending dashboards that track particle concentration, mean diameter, size distribution width (D90/D10), and zeta potential across multiple lots. Control charts flag deviations beyond predefined acceptance criteria (e.g., concentration CV >15%, diameter shift >10% from baseline), enabling early detection of process drift and supporting continuous process verification (CPV) programs.
Service Specifications & QC Standards
Instrumentation & Capability
- NTA: NanoSight NS300 or equivalent, 405 nm or 532 nm laser, sCMOS camera, NTA 3.4 software.
- TEM: JEM-1400 or equivalent, 80–120 kV, Gatan digital camera, minimum 10 fields of view per sample.
- Zeta Potential: Zetasizer Nano ZS or equivalent, PALS detection, disposable folded capillary cells.
- Sample volume per assay: NTA (50–100 μL), TEM (10–20 μL), Zeta (100–150 μL).
Typical Data Range
- Particle size detection range: 10–2000 nm (NTA); 1–100 nm resolution (TEM).
- Concentration range: 107–1011 particles/mL (NTA, sample-dependent).
- Zeta potential range: –100 to +100 mV; typical mEV values: –20 to –50 mV.
- Mean diameter precision: ±2 nm (NTA, monodisperse standards).
- Concentration precision: ±10% (NTA, calibrated polystyrene beads).
Turnaround Time
| Project Type |
Timeline |
| NTA only |
3–5 business days |
| TEM only |
5–7 business days |
| Zeta potential only |
2–3 business days |
| NTA + TEM + Zeta (standard package) |
7–10 business days |
| Batch QC trending (5+ batches) |
10–14 business days |
| Expedited analysis |
+50% fee, 50% time reduction |
Timeline may vary based on sample type, volume, and assay customization requirements.
Deliverables
- NTA: Concentration and size distribution report, AVI video files, CSV raw data.
- TEM: Digital micrographs (minimum 5 images), scale bars, morphology assessment.
- Zeta: Zeta potential distribution report, conductivity data, phase plot.
- Integrated report: Cross-modal summary with statistical analysis and QC flags.
- Certificate of Analysis (CoA) per batch.
Quality Control
- Daily instrument calibration with certified polystyrene bead standards (NTA: 100 nm; Zeta: –42 ± 4 mV standard).
- Negative controls (particle-free PBS) run with every batch.
- Positive controls (commercially available mEV standards where applicable).
- Inter-operator consistency checks for TEM imaging and particle counting.
- Compliance checklist for minimal characterization requirements aligned with industry guidelines for extracellular vesicle studies.
- Optional GxP-aligned assay validation and CQA trending analysis for lot-release documentation.
Sample Requirements
| Required Information |
Optional Information |
Not Accepted |
- Sample type (OMVs, CMVs, fungal EVs, phage vesicles)
- Culture supernatant or purified vesicle suspension
- Approximate particle concentration (if known)
- Sample volume (minimum 200 μL for full package)
- Buffer composition and pH
- Storage conditions and shipping temperature
|
- Prior purification method and buffer composition
- Target application (research, CMC, regulatory)
- Desired detection sensitivity or special parameters
- Reference batch for comparison studies
- Regulatory documentation requirements (CoA format)
|
- Samples in organic solvents or fixatives
- Samples with visible precipitation or aggregation
- Samples without proper cold-chain documentation
- Bacterial cultures without supernatant clarification
- Contaminated or mixed samples
- Samples shipped at room temperature
|
Recommended Sample Quantity by Assay:
| Assay |
Minimum Volume |
Recommended Volume |
| NTA only |
50 μL |
100 μL |
| TEM only |
10 μL |
20 μL |
| Zeta potential only |
100 μL |
200 μL |
| Full package (NTA + TEM + Zeta) |
200 μL |
500 μL |
| Batch consistency (triplicate) |
600 μL |
1.5 mL |
Storage & Shipping: Ship purified vesicle suspensions on wet ice (4°C) or dry ice (–80°C) with cold-chain documentation. Avoid repeated freeze-thaw cycles. Recommended buffer: sterile PBS or low-conductivity buffer for zeta measurements. Ship culture supernatants clarified by centrifugation (≥10,000 × g, 30 min) to remove cells and debris.
Our Advantages
- Microbial EV Analytics Specialization: Every protocol is optimized for mEVs, not adapted from mammalian exosome templates. We account for OMV lipid-A content, CMV peptidoglycan association, and fungal EV cell-wall debris that confound standard analytics.
- Integrated Three-Parameter Platform: NTA, TEM, and zeta potential are performed under harmonized sample preparation and data interpretation standards, eliminating inter-lab variability and reducing client coordination burden.
- Industry-Standard Minimal Characterization: Our standard package directly addresses minimal characterization requirements for extracellular vesicle studies (quantification, morphology, and at least one additional physicochemical property), supporting publication and regulatory compliance.
- Batch QC Trending & CPV Support: For manufacturing clients, we offer longitudinal QC dashboards with statistical process control, enabling continuous process verification and early detection of production drift.
- Regulatory-Ready Documentation: CoA, SOP summaries, instrument calibration records, and method validation data are prepared in formats compatible with IND-enabling CMC packages and lot-release documentation.
Applications
Vaccine Adjuvant & OMV Product Development
Quantitative particle profiling ensures OMV vaccine candidates meet lot-release specifications for particle concentration, size uniformity, and surface charge consistent with LPS-containing outer membrane composition.
Therapeutic Drug Delivery System QC
mEV-based nanocarriers require validated particle size and surface charge data to predict biodistribution, cellular uptake, and formulation stability during shelf-life studies.
Probiotic & Functional Food-Grade mEV Manufacturing
Food-grade mEVs from GRAS Bacillus subtilis, GRAS lactic acid bacteria, or QPS-listed strains require consistent particle characterization to support functional claims and safety documentation.
Cosmetic & Skin-Targeted Vesicle Ingredient Validation
Cosmetic mEV actives demand validated particle size (dermal penetration prediction), morphology (integrity confirmation), and zeta potential (formulation compatibility) for ingredient dossiers.
Case Study
Case Study: Ultrastructural and O-Glycoproteomic Characterization of Prevotella intermedia Outer Membrane Vesicles
Prevotella intermedia, a Gram-negative periodontal pathogen, produces outer membrane vesicles (OMVs) implicated in host-pathogen interactions. Prior to this study, comprehensive O-glycosylation and ultrastructural characterization of its OMVs remained unreported. Researchers combined cryo-electron tomography (cryoET) and LC-MS/MS glycoproteomics to establish the first O-glycoproteome baseline for this species.
CryoET revealed an electron-dense surface layer (EDSL) enveloping both cells and OMVs—a novel feature for Prevotella. OMVs frequently exceeded 200 nm and displayed two distinct populations: electron-dense and translucent lumens, indicating differential periplasmic protein loading. A single major O-glycan (dHex-dHex-HexNAc(HPO3-C6H12O5)-dHex-Hex-HexA-Hex(dHex), 1531.48 Da) was identified across 443 unique O-glycosylation sites within 224 glycoproteins, including 14 T9SS cargo proteins. The O-glycosylation motif was extended to D(S/T)(A/I/L/M/T/V/S/C/G/F/N/E/Q/D/P), broader than reported in other Bacteroidota species. This integrated multi-modal approach demonstrates the value of combining ultrastructural imaging with glycoproteomics for microbial EV profiling.
Figure 2. Cryo-electron tomography of Prevotella intermedia cells and OMVs. (Ye, et al. 2024)
FAQs
Q: What is the difference between NTA and DLS for particle sizing?
A: NTA tracks individual particles via Brownian motion, providing particle-by-particle size distribution and direct concentration counts. DLS measures bulk light scattering intensity, which is biased toward larger particles. For polydisperse mEV samples, NTA offers superior resolution and concentration accuracy.
Q: Can you analyze crude culture supernatant, or do samples need to be purified?
A: We strongly recommend purified vesicle suspensions for accurate NTA and TEM. Crude supernatant contains cells, debris, and protein aggregates that confound particle counting. If crude supernatant must be analyzed, we apply differential centrifugation as a pre-processing step (additional fee applies).
Q: What is the minimum particle concentration detectable by NTA?
A: The practical detection limit is typically 106–107 particles/mL for most mEV samples, depending on particle size and scattering properties. Below this concentration, particle tracking becomes statistically unreliable. We recommend concentrating samples by ultrafiltration if the expected concentration is low.
Q: How does zeta potential relate to mEV stability?
A: Zeta potential indicates the magnitude of electrostatic repulsion between particles. mEVs with zeta potential more negative than –30 mV typically exhibit good colloidal stability (resistance to aggregation). Values between –10 and +10 mV suggest high aggregation risk. Surface modifications (PEGylation, antibody conjugation) often shift zeta potential and are monitored by this assay.
Q: Can TEM distinguish OMVs from CMVs or fungal EVs?
A: Negative-stain TEM reveals morphology differences: OMVs typically appear as spherical vesicles (50–250 nm) with electron-dense membranes, though cup-shaped morphology may be observed as a dehydration artifact during sample preparation; CMVs from Gram-positive bacteria may show thicker membranes and variable shapes; fungal EVs often display heterogeneous sizes and cell-wall associated debris. However, definitive classification requires complementary biochemical marker analysis.
Q: Do you provide GxP-compliant or GLP-grade analysis?
A: Our standard service is research-grade (R&D) with validated methods and calibration protocols. GxP-aligned analysis (instrument qualification, method validation, audit trails) is available as a custom service with additional documentation and quality oversight.
Q: What is polydispersity index (PDI), and why does it matter?
A: PDI is a parameter derived from dynamic light scattering (DLS) that quantifies the width of the particle size distribution, ranging from 0 (monodisperse) to 1 (highly polydisperse). For mEV products, PDI <0.2 indicates a narrow, uniform population desirable for therapeutic applications. High PDI (>0.4) suggests sample heterogeneity, aggregation, or contamination. Note: NTA instruments report size distribution width rather than PDI.
Q: Can you compare my sample against a reference batch or competitor product?
A: Yes. We offer comparative analysis services that profile your sample side-by-side with a reference batch, commercial standard, or competitor product. Reports include statistical comparison of mean diameter, concentration, and zeta potential with significance testing.
References:
- Ye, X., et al. (2024). Ultrastructural and glycoproteomic characterization of Prevotella intermedia: Insights into O-glycosylation and outer membrane vesicles. Microbiologyopen, 13(2), e1401.
