Besides determining the number of extracellular vesicles (EVs) and the size per EV, would it be possible to create an ‘exosome profile’? This means characterizing molecular compositions of EVs and discriminating between different EV populations?

We suggest undertaking a feasibility study and testing a “good profile” versus a “bad (low-quality) profile” to find out if OF2i® provides the results the client is looking for. The molecular composition of the particles can be identified through their scattering behavior. However, we’ll need to conduct a feasibility test to determine if OF2i® can reliably make these differences visible for your specific samples. Before we make any statements, we would first need to know the following parameters: - What is the size range of the particles? - Do you probably know the reflective index of your particles? - Could you share any other details about your samples?

Can the sample be recovered after measurement?

Yes, the sample can be reused because the measurements are non-destructive.

Can we be sure that the data from the BRAVE facility is 100% consistent with the analysis data for the entire product?

We provide statistical values, which are representative of the entire product. This cannot be 100 % but the statistics give results close to 100 % (>99 %). What we recommend to our customers is that the device be installed for information purposes only at the beginning and that measurements are taken using their existing analysis methods at the same time for comparison. This makes it easy to determine whether the results meet the customer's specifications.

Can you identify PFAS in water?

Yes, we can detect and identify PFAS particles with the OF2i®-Raman device. However, it does depend somewhat on the size of the particle. The device can characterize the size, particle size distribution and particle concentration of particles from 100 nm to 150 nm. With OF2i®-Raman it is easier to detect PTFE than PFOS (although this is all PFAS). We are working on further use cases to define these limits.

Can you share some application examples and results? (products, application, object, test results, etc.)

Example measurement reports can be found on our homepage: https://braveanalytics.eu/en/of2i-en/ - please scroll down to section 5.

Does OF2i® provide absolute particle size measurement?

Please note: absolute particle size means that the size measured with a microscope matches the size measured with BRAVE Analytics’ device. Yes, OF2i® measures the absolute size of particles based on their hydrodynamic radius.

How long is the sample measurement time for OF2i®?

OF2i® lab device: the first results can be seen within seconds. In the settings you can decide when and how often you would like to have a report for your measurements. OF2i® PAT device: due to the sample bypass there is a time lag between taking sample and measurement. This is about 1 minute (depending on concentration and required dilution). You can decide when, for how long and how often you would like to have a detailed report of the measurement. The data can also be transferred directly into the computing system – usually MQTT interfaces are used for this.

How many particles need to be in the sample for OF2i® to detect and determine their size? How can you increase the sensitivity of the system?

The limits of the system are as follows: Typically OF2i® “sees” around 1 % of all the particles in the sample. On a sample with very small particles e.g. 200 nm the measuring parameters are set to 4 µl/min and this should enable a measurement of at least 100 to 1000 particles. If the particles are large (>3 µm) the measuring parameters can be set to 200 µl min.

If air bubbles are visible in the system (this will distort the laser shape) what is the problem?

It is possible that: the sample vial is empty a fitting is too loose a fitting is too tight the tubing is blocked To troubleshoot, check the sample vial and refill sample if needed. If the laser beam is not OK, perform a cell cleaning routine. A single air bubble may be stuck in the measuring cell. If the problem persists switch the laser off in the software (“Laser OFF” in the Global tab). See the instruction manual for more information.

If B-Curious can only measure particle sizes up to 3 or 5 µm, why can B-Elementary detect particles up to 50 µm (or more)? It is just the same OF2i® device plus a Raman spectrometer…

Each technology covers a certain size range and these size ranges are also sample-dependent. These are: OF2i® (B-Curious/ B-Continuous) is the basis for all other technologies and covers particle sizes between 50 nm and- 3 µm. You can extend this range by adding modules e.g.: B-Aware: size range 5 nm to 50 nm B-Elementary: particles identified in size rang 250 nm to 50 µm (Raman analysis) There is an overlap in the particles sizes up to 3 µm. Larger particles >3 µm can only be analyzed with the Raman module. We are working on extending the OF2i® size range.

If there are bubbles in the product (sample), will it affect the analysis? For example, if the product has a lot of bubbles due to homogenizing using a bottom mixer.

Bubbles are indeed a problem for our measurements. However, we assume that the product must be diluted before it is fed into the PAT device. During the dilution process, the bubbles can be reduced to a minimum.

Is it possible to track and analyze only specific particle sizes among the many particles passing through a flow cell? If so, how it can be done?

OF2i® measures the number-based size distribution within the size range 100 nm to 3000 nm. If the specific size is in this range, the requested specific size will be resolved and shown in a histogram. Please note: Other particle populations can be identified using the Raman module

Is there a temperature change when the sample passes through the flow cell? If the temperature change affects the sample, is there a mechanism to keep the temperature constant?

If the particles absorb light in the used wavelength of 532 nm, temperature change can happen (e.g. carbon nano tubes, certain ceramic particles, certain polymer particles). This is very sample-dependent and is usually not the case for biological samples. The temperature change within the cell is measured during the measurement, and if the temperature change is bigger than 1 °C, measurement values are corrected.

Mass spectroscopy coupling - how does this work? Who is interested?

Coupling OF2i®-Raman with mass spectroscopy is interesting for people who want to combine atomic resolution after first performing particle sizing and Raman analysis on a single-particle basis. ICP-MS is good at resolving heavy materials e.g. metals but is not good with plastics. OF2i®-Raman is good at determining/identifying plastics but not so good with metals. This makes the combination very interesting for scanning particles in environmental samples.

OF2i® PAT device: what is the delivery date and approximate price?

The approximate price depends on the customer requirements. Factors include: The sample Which parameters should be measured What has been used so far What is the customer’s problem A template is provided to help get a good case definition and decide which product would be the best fit. It includes questions to ask the potential customer. Download the template under DL > Materials > Templates The delivery date is usually 6 months after ordering the device + time for customs, etc.

OF2i® PAT device: what utilities does the customer need to provide?

BRAVE Analytics will set up the installation of the device as a project together with the customer because the hardware interface needs to be set up depending on the process. In principle, the following requirements/information must be taken into account:

OF2i® PAT device: Which product do you need for the test sample, downstream or upstream?

For some products the characteristics of the product may change depending on the surrounding temperature and time after certain processing steps, e.g. homogenization. This must be taken into account. Please provide detailed SOPs and dilution media with the “good” and “bad” samples for the stages which are relevant to your customer.

PAT OF2i® device: What does the cleaning process (CIP) look like?

The bypass to the PAT device and the tubes / cell inside the PAT device can be cleaned with different media (ethanol, etc.). The device itself will not be in the CIP environment; the sterile border is before the device. (More explanations can be provided when the customer’s setup and sample is defined).

Raman analysis module – do you need to know what you are looking for or can you read out spectra and access a database to find out what the particles are (non-target screening)?

At the moment we have a database of around 10 material spectra. Larger databases are available and the customer can search for the spectra in these databases. If necessary we can integrate a larger database (but this is not planned now).

Raman analysis module – why can‘t the device detect smaller particle sizes?

The Raman module does not detect particles smaller than 500 nm at the moment and we are working on developing a Surface-Enhanced Raman Spectroscopy (SERS) module for the analysis of smaller particles. Product launch approx. 2026-2027.

The customer wants to check the performance of the equipment. What are the references and standards?

During installation our technician will perform an IQ OQ PQ. We use standards for checking the precision of our measurements.

The OF2i®-Raman module detects and identifies single microplastic particles in water. Does that mean that the lower level of detection (LOD) is at around one particle per milliliter of sample? Are there also a certain number of particles passing by the analytical window without being detected?

If there is just one particle in one milliliter of e.g. water it is very difficult to predict the likelihood of detecting this one particle because the particle could pass by the detection window and go unseen. If the particle is larger i.e. Large Particle Counts in the micrometer range, it becomes more likely to detect the single particle. We are working on a module which looks at the whole measuring cell (capillary). This module will be available from mid-2025.

What can be ordered now and be delivered starting in Q3 2025?

1. BRAVE B-Phat (software + hardware module) for Large Particle Counts with ultra-low concentrations. Range: up to 50 micrometers 2. BRAVE B-Prepared (sofrware + hardware module) for automated sample preparation and automated dilution 3. BRAVE B-Elementary Tweezer

What can be ordered now and delivered within 6 months?

1. BRAVE B-Curious (our lab device for PSD & particle concentration. Range: 50 nm to 5000 nm) 2. BRAVE B-Aware (software module for understanding aggregation behavior of very small particles based on static light scattering models. Range: 5 nm to 50 nm) 3. BRAVE B-Elementary (software + hardware Raman module. Range: 500 nm to 50 micrometers) 4. Fluid automation module (software + hardware) for 8 samples per tray. Autosampler and automated self-cleaning.

What is the maximum particle size which can be measured with OF2i®?

The particle size determined by OF2i® can be max. 1/3 of the diameter of the capillary (to prevent clogging of the capillary).

What is your global delivery performance for the equipment?

For the deliveries we are using well-qualified transportation companies with a proven track record for overseas deliveries. In regards of the documentation the following documents are provided to the authorities: - Commercial invoice - Packing list - Certificate of origin - Customs declaration - Air waybill - CE conformity declaration - Insurance certificate

When are demos offered?

On-site demos and rentals / leasing are generally fine for us. In these cases the following questions need to be answered: - Before a demo: Who are the users and is there a willingness to pay after the demo (either a subsequent rental agreement and or high probability for purchase of a device)? - Possibility for a joint scientific project would also be an option. Here in Graz we have given discounts if the customer writes publications and/or presents us in the scientific community e.g. at project presentations or conferences.

Which sizes and which materials can be measured with OF2i®?

Using an OF2i® scan (particle sizes 100 nm to 3 µm) with some exceptions: biological material: ~150 nm to 3 µm anorganic materials: ~100 nm to 3 µm metal oxides: ~100 nm to 3 µm plastics and polymers: ~100 nm to 5 µm 2. In the range from ~50 nm to ~100 nm OF2i® estimates the number of objects as a percent of all the existing objects compared to the number of measured objects in the size range 100 nm to 3 µm. 3. Light scattering range (for high concentrations). This is not single-particle-based and is used for characterizing very small particles (e.g. proteins, e.g. 10 nm to 50 nm) at around 5 mg/ml. 4. Light obscuration range for determining the Large Particle Count, particles >5 µm at concentrations of <1000 objects / ml.

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As a partner in the NanoPAT project, BRAVE Analytics has received funding from the European Union’s HORIZON 2020 research and innovation programme under grant agreement n°862583.

As a partner in the MOZART project (MOZART for safe & sustainable by-design metallic coatings & engineered surfaces (RIA)), we received funding from the European Commission as part of the EU Horizon 2020 program.