Jun 16, 2026
Green Synthesis of Selenium Nanoparticles Using Rosemary Aqueous Extract and Ascorbic Acid
- Mohammadreza Ashayeri1
- 1Tarbiat Modares University
Protocol Citation: Mohammadreza Ashayeri 2026. Green Synthesis of Selenium Nanoparticles Using Rosemary Aqueous Extract and Ascorbic Acid. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbo1oolpk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 29, 2025
Last Modified: June 16, 2026
Protocol Integer ID: 236009
Keywords: Selenium nanoparticles, Green synthesis, Rosmarinus officinalis, Ascorbic acid, Biogenic nanoparticles, friendly synthesis of selenium nanoparticle, green synthesis of selenium nanoparticle, selenium nanoparticle, using sodium selenite, sodium selenite as the selenium precursor, nanoparticle formulation, selenium precursor, using rosemary aqueous extract, rosemary aqueous extract, nanomedicine research application, resulting nanoparticle, officinalis aqueous extract, ascorbic acid, ascorbic acid this protocol, green synthesis
Abstract
This protocol describes a reproducible, environmentally friendly synthesis of selenium nanoparticles (SeNPs) using sodium selenite as the selenium precursor, L-ascorbic acid as a reducing agent, and Rosmarinus officinalis aqueous extract as a natural reducing and capping agent. The method produces nanoparticle formulations: Se-Ros-NPs. Optimal conditions include controlled heating at 60 °C, dropwise addition of reducing agents, and a 48-hour incubation at 37 °C. The resulting nanoparticles are suitable for biological, physicochemical, and nanomedicine research applications.
Image Attribution
Figure 1: Graphical abstract illustrating green synthesis methods of selenium nanoparticles using Rosmarinus officinalis extract and ascorbic acid, typical characterization results (UV-Vis spectroscopy and DLS size distribution).
Materials
Reagents:
- Sodium selenite (Na₂SeO₃), ≥99%
- L-ascorbic acid, analytical grade
- Dried Rosmarinus officinalis leaves
- Deionized water (fresh, ≤48 h)
- Optional: NaCl (50 mM for purification aid)
Consumables:
- Whatman No.1 filter paper or 0.45 µm membrane filters
- 50 mL polypropylene centrifuge tubes
- Sterile glass reagent bottles
- Syringes (5–20 mL) for dropwise addition
Equipment:
- Hot plate with magnetic stirrer
- Magnetic stir bars
- Analytical balance
- pH meter or pH indicator strips
- Centrifuge (≥6000 rpm; preferable 10,000–12,000 rpm)
- Incubator (37 °C)
- Filtration apparatus
- Thermometer or digital temperature probe
Troubleshooting
Problem
Problem 1: No color change
Solution
Possible Causes:
- Ascorbic acid oxidized
- Rosemary extract degraded
- pH too low/high
Solutions:
- Prepare fresh AA
- Re-extract rosemary
- Adjust pH to 6.5–7.5
Problem
Problem 2: Large particle size (3e150 nm)
Solution
Possible Causes:
- Rapid addition of reducing agents
- Overheating ( 3e65 °C)
- Too high concentration of extract
Solutions:
- Add extract slowly ( 3e3 min)
- Maintain strict 60 °C
- Dilute extract 1:1
Problem
Problem 3: Aggregation during or after synthesis
Solution
Causes:
- Over-reduction
- Ionic contamination
Solutions:
- Lower AA concentration
- Use ultrapure water
- Limit reaction time
Problem
Problem 4: No pellet after centrifugation
Solution
Causes:
- Highly colloidal NPs
- Too low centrifuge speed
Solutions:
- Increase rpm to 12,000
- Add 50 mM NaCl to destabilize
Problem
Problem 5: Extremely dark brown color
Solution
Cause:
- Excess reductant or too rapid reduction
Solution:
- Slow down dropwise addition to ≥5 min
Safety warnings
Critical Notes:
- Do NOT exceed 60 °C (phenolics degrade).
- Darker extract indicates higher phenolic content; may cause faster reduction.
Safety Notes
- Sodium selenite is toxic and should be handled in a fume hood.
- Wear gloves, mask, and goggles.
- Dispose of selenium waste separately as a hazardous heavy metal.
- Avoid inhalation of rosemary powder during grinding.
Before start
Preparation of 1% (w/v) Rosemary Extract
- Weigh 1 g dried rosemary leaves and grind to fine powder.
- Add to 100 mL fresh deionized water to a beaker.
- Heat to 40 °C and stir at 1800 rpm for 60 minutes.
- Transfer to a centrifuge tube and centrifuge at 6000 rpm for 30 minutes.
- Collect the supernatant.
- Filter through Whatman No.1 OR 0.45 µm membrane.
- Store at 4 °C (use within 24–48 hours).
Graphical abstract illustrating green synthesis methods of selenium nanoparticles using Rosmarinus officinalis extract and ascorbic acid, typical characterization results (UV-Vis spectroscopy and DLS size distribution).
Preparation of 1% (w/v) Rosemary Extract
Weigh 1 g dried rosemary leaves and grind to fine powder.
Add to 100 mL fresh deionized water to a beaker.
Heat to 40 °C and stir at 1800 rpm for 60 minutes.
Transfer to a centrifuge tube and centrifuge at 6000 rpm for 30 minutes.
Collect the supernatant.
Filter through Whatman No.1 OR 0.45 µm membrane.
Store at 4 °C (use within 24–48 hours).
Preparation of Stock Solutions
Dissolve 0.263 g Na₂SeO₃ in 100 mL DI water.
Dissolve 0.176 g ascorbic acid in 100 mL DI water.
Prepare fresh; sensitive to oxidation.
SeNPs Synthesis Procedures
Heat 20 mL Na₂SeO₃ to 60 °C.
Add 20 mL rosemary extract dropwise over 2–4 minutes while stirring, then continue stirring for 30 minutes total.
Add 20 mL 10 mM ascorbic acid dropwise over 3–5 minutes while stirring, then continue stirring for 30 minutes total.
Incubate at 37 °C for 48 hours.
Purification
Centrifuge at 8000 rpm for 20 minutes.
Discard supernatant.
Resuspend pellet in DI water.
Repeat 2–3 times.
Rosemary-capped NPs may require 10–12k rpm.
If no pellet forms, add 50 mM NaCl to reduce colloidal stability.
Characterization
UV–Vis: Expected peak: 260–300 nm.
DLS: Expected size: 45–90 nm depending on formulation.
Zeta Potential: Se-AA-Ros: approx. –15 mV
XRD: Match with JCPDS 06-0362 (trigonal Se)
FTIR: O–H stretching (3300 cm^^–1^^)
C=O (1730 cm^^–1^^)
Phenolic groups ~1600 cm^^–1^^
Troubleshooting
Problem 1: No color change
Possible Causes:
- Ascorbic acid oxidized
- Rosemary extract degraded
- pH too low/high
Solutions:
- Prepare fresh AA
- Re-extract rosemary
- Adjust pH to 6.5–7.5
Problem 2: Large particle size (>150 nm)
Possible Causes:
- Rapid addition of reducing agents
- Overheating (>65 °C)
- Too high concentration of extract
Solutions:
- Add extract slowly (>3 min)
- Maintain strict 60 °C
- Dilute extract 1:1
Problem 3: Aggregation during or after synthesis
Causes:
- Over-reduction
- Ionic contamination
Solutions:
- Lower AA concentration
- Use ultrapure water
- Limit reaction time
Problem 4: No pellet after centrifugation
Causes:
- Highly colloidal NPs
- Too low centrifuge speed
Solutions:
- Increase rpm to 12,000
- Add 50 mM NaCl to destabilize
Problem 5: Extremely dark brown color
Cause:
- Excess reductant or too rapid reduction
Solution:
- Slow down dropwise addition to ≥5 min
Safety Notes
- Sodium selenite is toxic and should be handled in a fume hood.
- Wear gloves, mask, and goggles.
- Dispose of selenium waste separately as a hazardous heavy metal.
- Avoid inhalation of rosemary powder during grinding.
Expected Results
Formation of stable SeNPs with red/orange coloration.
Particle size: 45–90 nm.
Negative zeta potential (–5 to –15 mV) ensures stability.
Stable dispersions for up to 4 weeks at 4 °C.
Rosemary extract improves capping efficiency and nanoparticle uniformity.