TITLE: Microwave Synthesis of Lanthanum-Doped Carbon Dots
AUTHORS: Samuel Holberg, Addy Cullen, Chih-Yu (Jill) Lee, Destiny Caulder, Geisianny AM Moreira, Eric S McLamore

[DESCRIPTION]
This protocol describes the synthesis of lanthanum-doped carbon dots using microwave irradiation. The complete process requires approximately 1h 50 min to complete (including baseline analysis). For this protocol, an Anton-Parr microwave reactor (Monowave 50p) was used throughout. The application example is for water quality sensing, although we have used the particles for cell imaging in other studies. ADA compliant procedures are used throughout the protocol (including both building lab space/design and analysis of synthesized particles).

[BEFORE_START]
Be sure to wear appropriate safety PPE throughout (lab coat, gloves, eyewear).
Electronic or physical lab notebook may be used throughout
See experimental plan guide for tips on planning your work

[GUIDELINES]
This protocol describes the synthesis of lanthanum-doped carbon dots using microwave irradiation. The complete process requires approximately 1h 50m to complete (see diagram for overview of steps). 

A link to the Monowave Resource Manual with additional basic information on the instrument is here (link).
    


[STEPS]
SECTION: SECTION 1) Preparation
1. Prepare Solution 1 (200 mM D-glucose)
Prepare a sealed plastic or glass container that has at least 10 mL of capacity and label as 200mM D-glucose
Weigh 0.721 g of D-glucose and solvate in 200 mL of DI/nano-pure water
Mix solution and look carefully to ensure there are no precipitates
Cover with Parafilm
Heat in microwave for 90 s to expedite solvation
Shake/stir well and look carefully to ensure there are no precipitates
Seal vial immediately (contamination is a common problem)
SECTION: SECTION 1) Preparation
5. Prepare reaction vials

Check vial cap and Teflon liner to make sure neither is damaged, and both are clean (Fig 2A-B)

 

 

 
SECTION: SECTION 1) Preparation
2. Prepare Solution 2 (2.5 mM KCl)
Prepare a sealed container with 1L capacity and label as 2.5 mM KCl
Weigh 0.1864 g and solvate in 1 L of DI/nano-pure water
Mix and inspect carefully to ensure there are no precipitates
Seal vial
SECTION: SECTION 1) Preparation
3. Prepare Solution 3: (100mM MES buffer)
Prepare a sealed plastic or glass container that has at least 1L of capacity and label as 100mM MES buffer
Prepare a solution of 0.1 Molarity (m) 2-(N-morpholino)ethanesulfonic acid (MES buffer). This buffer has a pKa of 6.15 at 25°C; MW=195.2 g/mol (link to Good’s buffers)
For a 1 L buffer solution, weigh 19.52 g of MES in a plastic weigh boat 
Transfer the MES powder from the weigh boat to the labeled storage container
Add 1 L of DI water or nano-pure water
Mix solution and look carefully to ensure there are no precipitates
Check the pH of the solution (should be approximately pH 6.2). 
Seal vial
SECTION: SECTION 1) Preparation
4. Prepare solution 4 (200mM La2CO3)
Prepare a sealed plastic or glass container that has 9.9mL of MES buffer and label as MES+200mM La2CO3
Prepare solution of 6 Molarity (M) HCl (see guidance here)
 

Weigh 0.916 g of La2CO3 in weigh boat capable of holding 1mL of fluid
Transfer the weigh boat and container to the chemical hood
Pipette 0.1 mL of HCl (6M) into the petri dish and carefully agitate the dish until all powder is dissolved
Carefully transfer the solvated La2CO3 solution to the container
Mix solution and look carefully to ensure there are no precipitates
Calculate the theoretical pH according to Henderson/Haselbalch equation:

   
Check the pH of the solution (should be approximately pH 5.5), or within 2% of calculation based on pKa
Seal vial



SECTION: SECTION 1) Preparation
6. Add solution to reaction vial

Pipette 3 mL of 0.2 Molarity (m) D-glucose solution to a glass reactor vial
Add a nano stir bar to the reactor vial
Add 3 mL of the La2Co3 + buffer solution to the reactor vial
Place Teflon liner in cap
Seal reaction vial with cap
Invert vial three times to mix solutions
Place in test tube rack

 

SECTION: SECTION 2) Particle synthesis
7. Set up microwave reactor

Ensure that Anton-Paar reactor is in chemical hood and plugged in to the power outlet (Fig 3A)
If not placed in the chemical hood, the institutional safety committee must approve of the exhaust hose setup (using either a snorkel exhaust and/or valid tube/hose system)
Open lid and place glass reactor in Anton-Paar reactor chamber (Fig 3B)
Close chamber lid and seal shut with handle

  
Select LaCD settings in the “Run by Methods” (Fig 4) by pressing the Menu button
Press Menu
Press Methods button 
Choose LACD1.1
Click Done
Click back
 

Choose Run Name
In the text box, name the file as “LACD-today’s date” (Fig 4)
Prior to starting the Monowave, make sure that the settings are correct and have not been changed
Maximum set temperature= 170 °C
Time to ramp setting= 5 min
Hold time= 1 min

  
Click run
This protocol is similar to other lanthanum-doped carbon dots in the literature and has a variety of uses from cell labeling to water quality analysis (REF 1-3).
SECTION: SECTION 4) Stabilization and analysis (post cook)
9. Stabilize particles in buffer
Prepare a sealed plastic or glass container that has 10 mL of capacity and label (Lanthanum-doped carbon dots + KCl). 
 


Mix the 6mL of pyrolyzed solution with 4 mL of 0.0025 Molarity (m) KCl. This results in a final concentration of 1mM KCl + LACD
 

Inspect solution carefully for any colloids or particulate. 
Seal vial 



SECTION: SECTION 3) Reactor data collection
8. Download reactor temp/pressure profile (Timing: 5 minute)

Insert USB to port on right side of Monowave 50 p
Press “Details”
Press “Export”
Choose the Export option, press “OK” when done
Remove USB
SECTION: SECTION 5) Cleanup
13. Clean up space and dispose of waste
Turn off Monowave and unplug it from power outlet
Dispose of used chemicals according to the lab safety plan


 


Wash all glass reactors with mild detergent and warm water. A bottle brush may be required to remove carbon residue from the inner wall of glass vials
Carefully remove the Teflon lining from the cap and wash with mild detergent and warm water
oIf the cap or the Teflon lining is damaged, they should be discarded according to the lab safety plan.
 

SECTION: SECTION 6) Data management
14. Data management (reactor data only)

File naming: For saving all versions of this protocol, use the following file structure:
            LACD1.1_(insert date here).ai

File storage: Store all methods in the Desktop folder.

Backup files: At least once per year, ensure that the folder is backed up on the lab external hard drive.

Note: data management for spectroscopy data is covered in a separate protocol.
SECTION: SECTION 4) Stabilization and analysis (post cook)
10. Qualitative analysis (color)

The first step of qualitative analysis is visual inspection of the solution color
Analysis of polychromatic visible (VIS) color is an important quality control mechanism in particle synthesis (Fig 5) 

  
 

Observe the glass vials under white light (common lab lighting)
Representative photos of "as prepared" particle solutions are shown in Fig 6.
The images depict a “burned sample” (Fig 6A), “undercooked sample” (Fig 6B) and an optimum sample (Fig 6C)
In addition to visual inspection, UV-VIS may be used to analyze the samples
  
Representative results of using a mobile phone app for identifying color are shown below (Fig 7).
The same photos as shown in Fig 6 are analyzed using an iPhone app, providing RGB and CMYK color space data for the identified color.

  

SECTION: SECTION 4) Stabilization and analysis (post cook)
11. Qualitative analysis (fluorescence emission)
Using either low light conditions or a prepared light box for analysis (e.g., black styrofoam), position a 395 nm UV pen perpendicular to glass storage vial, 
Turn on pen and inspect for an emission beam
Fig 8A shows an example of fluorescence for a sample following the protocol described here
This rapid analysis is useful for quality control immediately after synthesis of particles, but should be supported by subsequent quantitative analysis (shown in step below).
  
SECTION: SECTION 4) Stabilization and analysis (post cook)
12. Quantitative analysis (fluorescence emission)
If available, use a spectroscopy instrument to analyze the emission spectra under UV excitation
Fig 9 shows a representative experiment with a 370nm excitation source (fiber optic spectrometer from Ocean Optics; Ocean HR2 UV-VIS)
The figure shows changing fluorescence emission during addition of a quenching  molecule
Three peaks are identified (denoted as λm1, λm2, λm3), each with a different characteristic response to increasing concentration of the quencher molecule. 
  
When analysis is complete, place LACD in dark for up to 2 weeks (shelf life currently unknown; 2 weeks is conservative estimate)