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There is no universal sample-preparation recipe for label-free particle detection. The correct steps depend on the measurement method (such as dynamic light scattering or nanoparticle tracking analysis), particle material and size, sample matrix, instrument, and whether you need a size distribution, a particle count, or another property. Dilution, dispersion, filtration, centrifugation, settling, and sonication can all change the fraction that is measured. A defensible workflow therefore starts with representative sampling, uses a clean and compatible diluent, adjusts concentration for the specific instrument, and records every handling step.
Why preparation is part of the measurement
Label-free methods measure physical signals from particles rather than a fluorescent or chemical label. Preparation changes those signals and can change the particles themselves. A filter may remove dust but also retain target particles; centrifugation may reduce large interferents while losing a rapidly sedimenting fraction; sonication may break weak aggregates or create new ones; and dilution may shift an equilibrium between dispersed and aggregated states.
Sampling is therefore as important as instrument operation. In its regulated food and feed guidance, the European Food Safety Authority (EFSA, 2021) says sampling and preparation should represent the small-particle fraction as closely as possible and be appropriate to the analytical method. ISO/TS 24672:2023 likewise covers several number-concentration methods rather than prescribing one protocol for all samples. The protocol should state what was done, not simply say that a sample was “prepared.”
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Choose preparation from the method and the question
| Method | What it measures | Preparation priorities | Main interpretation risk |
|---|---|---|---|
| Dynamic light scattering (DLS) | Ensemble fluctuations in scattered light, converted to a diffusion coefficient and hydrodynamic size. | Clean, compatible diluent; dust-free labware; concentration adjusted to particle scattering; optional filtration or centrifugation only after checking analyte compatibility. | Intensity weighting makes large scatterers disproportionately influential, so a small amount of dust or a few aggregates can dominate the result. |
| Nanoparticle tracking analysis (NTA) or particle tracking analysis (PTA) | Brownian motion of individually visible particles, used to estimate size and concentration. | Dilution to the instrument’s validated particle-density range; a diluent-only blank; assessment of sedimentation and visibility. | Counts depend on camera, threshold, focus, settings, and preparation. Particles in the diluent can be counted as sample particles. |
| Other number-concentration methods | Depending on the method: particle size, number, mass, optical response, or another property. ISO/TS 24672:2023 discusses DCS, MDLS, SAXS, UV-vis, PTA, resistive-pulse sensing, spICP-MS, CPC, and DMAS. | Follow the validated method and instrument instructions for that sample form; do not transfer a DLS or NTA protocol automatically. | These methods do not measure the same particle property or necessarily the same fraction of a sample. |
Before preparing anything, define the output: hydrodynamic size, number concentration, concentration by another definition, or evidence of aggregation. Also record the particle-size range, chemistry, matrix, expected concentration, and whether aggregates are part of the question. NIST maintains separate nanoparticle dispersion preparation protocols because these details materially affect the procedure.
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A reproducible preparation workflow
1. Confirm the instrument and validated sample type
Check the instrument’s sample volume, optical limits, concentration window, cuvette or cell format, and compatible buffers. A method’s nominal operating range is platform-specific. For example, the OSTR Biophysics Core at the National Cancer Institute gives facility-specific DLS and NTA instructions; those values should not be treated as universal limits for every instrument.
2. Take a representative aliquot
Mix or subsample according to the material’s stability. Use gentle inversion when vigorous agitation could fragment particles or alter weak aggregates. If settling occurs during handling, define a consistent mixing and sampling time. Do not assume that sonication is harmless: it can disperse a real aggregate population, heat the sample, or damage soft particles.
3. Prepare and inspect the diluent
Use a diluent that is chemically compatible with the particles and does not dissolve, swell, precipitate, or destabilize them. For DLS, the NCI Nanotechnology Characterization Laboratory recommends a freshly filtered buffer and dust-free labware. For NTA/PTA, measure the diluent alone when background particles could be detected. A blank establishes whether the buffer, water, cell, or handling environment contributes to the count.
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Prepare a dilution series when the starting concentration is uncertain. DLS needs enough scattering for a stable correlation function but can be impaired by excessive scattering, multiple scattering, or large contaminants. NTA needs particles sufficiently separated for individual tracking while still providing enough tracks for a reliable estimate. Use the instrument’s recommended range as a starting point, then verify signal quality and repeatability at more than one dilution if practical.
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One attributable NTA example is the OSTR Biophysics Core’s typical stock dilution of 500–1,000 times to a final concentration of approximately 107 particles/mL for its stated facility and instrument. This is an instrument-specific example, not a universal NTA threshold.
5. Decide whether to remove larger material
Inspect whether visible dust, bubbles, or rapidly sedimenting material is interfering. A cleanup step is justified only when its effect on the target is understood. A filter can remove the particles you intend to count; centrifugation can selectively deplete dense or large particles; and allowing a sample to settle changes the measured fraction. If you remove a fraction, describe that choice rather than presenting the result as the composition of the original bulk sample.
6. Equilibrate and load consistently
Use the same delay between dilution, mixing, temperature equilibration, and measurement for every replicate. Load cells without introducing bubbles, fingerprints, or dust. Follow the instrument’s required fill volume and orientation. If particles sediment during the run, keep acquisition time and sampling position consistent or document the time-dependent change.
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7. Record the complete preparation
At minimum, record the original matrix, aliquot procedure, diluent composition and lot, dilution factor, mixing or sonication conditions, filtration membrane and pore size, centrifugation speed/time/temperature, settling time, storage and equilibration conditions, cell type, instrument model, and analysis settings. Published NTA work has found systematic differences associated with preparation and settings, so these details are necessary for comparison.
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DLS-specific preparation
Control contamination before it controls the result
DLS is highly sensitive to rare large scatterers because its output is intensity weighted. Use clean vessels, caps, pipette tips, and a compatible diluent. Handle open samples briefly and avoid dusty work areas. Run a diluent blank when contamination is plausible; an unexpectedly strong or unstable blank is a reason to clean the workflow before interpreting the sample.
Match concentration to scattering, not just particle count
The useful concentration depends on particle size, refractive index, optical properties, and instrument geometry. A concentration that works for one material may be too weak or too strong for another. If the correlation function is noisy at low concentration, concentrate only if that will not change the dispersion. If intense or unstable scattering appears at high concentration, dilute and check whether the apparent size distribution changes.
Filter only when the membrane is compatible
The NCI protocol identifies detergent-free 0.2 µm polyethersulfone as one possible low-protein-binding choice for many aqueous applications. That is an option, not a default instruction. Select membrane chemistry and pore size so the principal analyte is not adsorbed, retained, dissolved, or otherwise modified. Pre-rinse only when the method validates that step, and include the filtration procedure in the report.
Use the correct cell and volume
DLS instruments may require disposable cuvettes, quartz cells, or specialized low-volume cells. The OSTR Biophysics Core documents cell formats and sample volumes for its DynaPro NanoStar II and Mobius systems; another instrument may require different hardware. A compatible cell that is clean and free of scratches is part of sample preparation, not an interchangeable accessory.
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NTA and PTA-specific preparation
Establish a particle-free baseline
Measure the diluent blank under the same camera and analysis settings used for samples when feasible. Water, buffer salts, containers, and carryover from a previous sample can produce detectable particles. The blank result should be considered when deciding whether a low sample concentration is real or background-limited.
Keep particles trackable
Tracking requires a density at which individual particles can be resolved. Overcrowding produces overlapping trajectories; too few particles increases uncertainty. Dilute stepwise and check the image, track count, and concentration stability rather than relying on a single nominal factor. Camera level, detection threshold, focus, and analysis settings can shift the reported distribution, so hold them constant across replicates and comparisons.
Assess sedimentation before removing it
Large particles may settle during preparation or acquisition. Removing them by decanting, low-speed centrifugation, filtration, or a timed settling step produces a measurement of the remaining suspension, not necessarily the original sample. If the purpose is to characterize the stable supernatant, define that fraction and report the separation time and method. If the purpose is total particle concentration, such removal may answer the wrong question.
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| Action | Potential benefit | Possible bias | When to use |
|---|---|---|---|
| Filtration | Reduces dust or oversized material. | Retains target particles, causes adsorption, changes aggregates, or contributes extractables. | Only with a validated membrane and pore size for the matrix and analyte. |
| Centrifugation | Separates rapidly sedimenting debris or defines a supernatant fraction. | Depletes dense target particles and changes the size or concentration distribution. | When the fraction of interest is explicitly defined and recovery is understood. |
| Settling | Allows large material or bubbles to leave the measurement zone. | Time-dependent loss of a particle fraction; results depend on the waiting period. | When settling is part of the intended sample definition and timing is controlled. |
| Sonication or vigorous mixing | May disperse reversible agglomerates. | May fragment particles, alter soft materials, heat the sample, or create an artificial distribution. | Only under controlled energy, temperature, and duration appropriate to the material. |
Matrix and sample-form decisions
Aqueous nanoparticle dispersions
Control pH, ionic strength, surfactants, and protein content because each can alter aggregation and hydrodynamic size. Use a diluent that reproduces the intended chemical environment unless the method specifically calls for a different medium.
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Protein-rich or biological samples
Expect matrix components to scatter, adsorb to particles, or form their own particles. A buffer blank may not be sufficient if the matrix itself contributes background. Define whether the measurement concerns native particles in the matrix or an isolated fraction; separation steps can answer those questions differently.
Food, feed, powders and other complex materials
Extraction and dispersion can create a suspension that was not present in the original material. EFSA’s 2021 guidance emphasizes method-appropriate preparation and representative sampling for small particles in regulated food and feed applications. For dry powders, aerosols, or nonaqueous matrices, use a protocol validated for that sample form rather than adapting an aqueous nanoparticle recipe.
Common failure modes and fixes
| Symptom | Likely cause | Practical check |
|---|---|---|
| DLS size jumps between repeats | Dust, a few aggregates, concentration drift, or inconsistent mixing. | Run a diluent blank, inspect cells, prepare a dilution series, and standardize the mixing-to-measurement interval. |
| DLS gives an unexpectedly large size | Intensity dominance by rare large scatterers. | Check for visible contamination and compare an untreated aliquot with a justified cleanup step without assuming the cleaned result represents the original sample. |
| NTA count is high in the blank | Particles in diluent, cells, tubing, or carryover. | Replace or refilter diluent, clean or change the cell, and repeat the blank before measuring samples. |
| NTA tracks overlap or concentration is unstable | Particle density is outside the instrument’s useful range or focus/settings vary. | Dilute stepwise, keep camera and threshold settings fixed, and verify concentration across replicate videos. |
| Replicates disagree after a delay | Sedimentation, aggregation, evaporation, or temperature change. | Control equilibration and acquisition times, mix using a defined procedure, and report any time-dependent behavior. |
How to report a label-free particle measurement
- Identify the method, instrument model, cell or cuvette, and measurement objective.
- Describe the original sample, representative-sampling procedure, storage history, and temperature.
- State diluent composition, filtration of the diluent, dilution factor, and final concentration estimate.
- Report filtration, centrifugation, settling, sonication, mixing, and equilibration conditions, including what fraction was retained.
- For NTA/PTA, report blank results and camera, threshold, focus, video, and analysis settings.
- For DLS, state how concentration was selected and whether results are intensity-, volume-, or number-transformed.
- Explain any step that could remove, dissolve, fragment, or aggregate particles, and avoid comparing results made with materially different preparation workflows.
Standards and method selection
ISO/TS 24672:2023 is a useful map of nanoparticle number-concentration approaches, but it does not make DLS, NTA, resistive-pulse sensing, spICP-MS, SAXS, optical particle counting, and other methods interchangeable. NIST’s sample-preparation index and instrument-specific facility protocols are better starting points for a concrete procedure. Manufacturer guidance, such as the dilution and sedimentation discussions in NTA methodology documents, should be treated as platform guidance and verified for the instrument and matrix in use.
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