Evaluation of Fluorescent Extracellular Vesicles (EV) Reference Materials Using NanoSight Pro for Biomarker Detection

Introduction

The NanoSight Pro is a powerful tool used to measure the size and number of tiny particles, like Extracellular Vesicles (EVs), which are important in many areas of biological and medical research. While the system is well known for its ability to measure particle size and concentration, its advanced fluorescence capabilities are sometimes overlooked.

Fluorescence measurements can provide valuable information about what EVs are made of and what roles they play in the body—especially when studying specific biomarkers. However, detecting these biomarkers can be tricky, particularly when they are present in very small amounts. That’s why having reliable fluorescent reference materials is so helpful. These materials can be used to test how well the instrument is working, show that fluorescence can be used to detect biomarkers, and help researchers find the best ways to label their samples.

Here we present an overview of the Vesi-Ref CD63 GFP and Vesi-Ref CD63 mGN reference materials and their characterization using the NanoSight Pro system.

Introduction

Here we present an overview of the Vesi-Ref CD63 GFP and Vesi-Ref CD63 mGN reference materials and their characterization using the NanoSight Pro system.

Materials and equipment

Two reference materials were provided by Vesiculab: VesiRef-CD63-GFP and VesiRef-CD63-mNG. These are extracellular vesicle (EV)-based formulations enriched in CD63, a tetraspanin commonly used as an EV biomarker. Both materials are bioengineered to express either GFP (Green Fluorescent Protein) or mNG (mNeonGreen) in the EV’s lumen in the presence of the tetraspainin CD63 [1].

Both proteins have fluorescence properties when excited at 488 nm and emit light above 500 nm, making them ideal for analysis with the NanoSight Pro system, which is equipped with a 488 nm laser, a 500LP fluorescence filter, and NS Xplorer 1.2 software.

Sample Preparation and Analysis

Sample Preparation: Vesi-Ref CD63-GFP is provided in a lyophilized (freeze-dried) form.
- Add 100 µL of particle-free deionized (DI) water to the vial.
- Mix using a pipette, then vortex to ensure complete resuspension.
- Dilute the resuspended sample approximately 165-fold with particle-free DI water before analysis.
Vesi-Ref CD63-mNG is supplied as a liquid suspension
- Simply dilute the sample by approximately 165-fold using particle-free DI water before analysis.

Note: For accurate dilution and concentration measurements, use a gravimetric method (weighing the sample and diluent) rather than relying solely on pipette volumes.

NanoSight Pro Analysis

Analysis was performed using NanoSight Pro equipped with a 488 nm laser module, 500 nm long-pass fluorescence filter (500LP), and NS Xplorer 1.2 software. Both light scatter and fluorescence modes were used.

Measurement setup:

10 videos, each 150 frames
Flow rate: 5 µL/min (slow, continuous flow)
Three aliquots per sample, measured under identical conditions; results represent the average of three measurements.

Camera settings:

Light Scatter: Exposure 16–17 ms; Contrast Gain 4–6
Fluorescence: Exposure 40 ms; Contrast Gain 15.8

Results

Vesi-Ref CD63 GFP

Vesi-Ref CD63 GFP sample showed a high level of polydispersity, which is typical for extracellular vesicle samples. The size distributions obtained from both light scatter and fluorescence measurements were consistent (Figure 1), with a modal size in the range of 80-90 nm, a common size for small biologics. The average particle concentration measured in light scatter mode was 7.5 × 10¹⁰ particles/mL, while fluorescence mode reported 4.8 × 10¹⁰ particles/mL in fluorescence. This indicates that approximately 63% of the EVs were detected with GFP tag, suggesting effective labelling of EV population (Table 1).

The fluorescence signal was good, allowing for clear detection of the labeled particles. However, photobleaching—a gradual loss of fluorescence signal over time—was observed during the measurements, which is a common characteristic of fluorescent dyes (Figure 2).

The NS Xplorer 1.2 software, operating in fluorescence mode, successfully detected and analyzed the fluorescently labeled EVs. The size distribution from both light scatter and fluorescence measurements aligned well, further confirming the suitability of the NanoSight Pro system for analyzing fluorescent EV reference material.

[AN251216-figure1.png] AN251216-figure1.png — **Figure 1.** The NS Xplorer average raw size distribution with standard errors of Vesi-Ref CD63 GFP in light scatter (dark blue line) and fluorescence (orange line) with example of direct NTA images observed under light scatter and fluorescence mode.

**Table 1.** Vesi-Ref CD63 GFP table comparing main analysis parameters in the light scatter and fluorescence.
	Modes [nm]	Mean [nm]	D10 [nm]	D50 [nm]	D90 [nm]	Concentration [particles/ml]	Fluorescence Efficiency [%]
Light scatter	76.3	133	55	101	238	7.5E+10	63%
Fluorescence	78.8	125	48	94	228	4.8E+10	63%

[AN251216-figure2.png] AN251216-figure2.png — **Figure 2.** The NS Xplorer decay graph for VesiRef CD63 GFP sample illustrating the photobleaching properties of the sample.

Vesi-Ref CD63-mNG

The Vesi-Ref CD63-mNG sample also demonstrated a high-quality size distribution, with a modal size between 81-83 nm, consistent with expectations for extracellular vesicles (EVs). The size profiles obtained from light scatter and fluorescence measurements were closely aligned, indicating consistent detection across both modes (Figure 4).

The average particle concentration measured in light scatter mode was 6.1 × 10¹⁰ particles/mL, while fluorescence mode reported 5.1 × 10¹⁰ particles/mL. This corresponds to an estimated fluorescence detection efficiency of approximately 80%, suggesting a high proportion of EVs were successfully with the mNeonGreen (mNG) fluorophore (Table 2).

The fluorescence signal was strong and stable, allowing for clear and consistent detection of labeled particles. Although photobleaching was observed during the measurement process, the mNG signal remained brighter and more robust compared to GFP, supporting its suitability for fluorescence-based EV analysis.

As with the GFP-labeled sample, the NS Xplorer 1.2 software effectively captured and analyzed the fluorescent signal from the Vesi-Ref CD63-mNG sample. The close match in size distributions between light scatter and fluorescence modes further confirms the reliability of the NanoSight Pro system for analyzing this reference material (Figure 3).

[AN251216-figure3.png] AN251216-figure3.png — **Figure 3.** The NS Xplorer average raw size distribution with standard errors of Vesi-Ref CD63 mNG in light scatter (dark blue line) and fluorescence (orange line) with example of direct NTA images observed under light scatter and fluorescence mode.

**Table 2.** Vesi-Ref CD63 mNG table comparing main analysis parameters in the light scatter and fluorescence.
	Modes [nm]	Mean [nm]	D10 [nm]	D50 [nm]	D90 [nm]	Concentration [particles/ml]	Fluorescence Efficiency [%]
Light scatter	82.7	119	58	104	190	6.2E+10	82%
Fluorescence	81	114	53	98	188	5.1E+10	82%

Summary

The Vesi-Ref CD63-GFP and Vesi-Ref CD63-mNG reference materials are well-suited for evaluating the fluorescence capabilities of the NanoSight Pro Nanoparticle Tracking Analysis (NTA) system. Both samples are easy to prepare and contain high concentrations of CD63-enriched extracellular vesicles (EVs) labeled with bright, well-characterized fluorescent dyes.

Their strong fluorescence signals, combined with consistent size distributions between light scatter and fluorescence modes, make them ideal for assessing system performance in fluorescence mode. These materials provide a reliable and convenient way to test instrument sensitivity and optimize experimental conditions for biomarker detection in EV research.

[1] Wiklander, O.P.B., Mamand, D.R., Mohammad, D.K. et al. Antibody-displaying extracellular vesicles for targeted cancer therapy. Nat. Biomed. Eng 8, 1453–1468 (2024)