Microwave Synthesis of Lanthanum-Doped Carbon Dots

Prepare Solution 1 (200 mM D-glucose)

• Prepare a sealed plastic or glass container that has at least 10mL of capacity and label as 200mM D-glucose
• Weigh 0.721g of D-glucose and solvate in 200mL of DI/nano-pure water
• Mix solution and look carefully to ensure there are no precipitates
• Cover with Parafilm
• Heat in microwave for 0h 1m 30s to expedite solvation
• Shake/stir well and look carefully to ensure there are no precipitates
• Seal vial immediately (contamination is a common problem)

Prepare Solution 2 (2.5 mM KCl)

• Prepare a sealed container with 1L capacity and label as 2.5 mM KCl
• Weigh 0.1864g and solvate in 1L of DI/nano-pure water
• Mix and inspect carefully to ensure there are no precipitates
• Seal vial

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 0.1Molarity (M) 2-(N-morpholino)ethanesulfonic acid (MES buffer). This buffer has a pK_a of 6.15 at 25°C; MW=195.2 g/mol (link to Good’s buffers)
• For a 1L buffer solution, weigh 19.52g of MES in a plastic weigh boat
• Transfer the MES powder from the weigh boat to the labeled storage container
• Add 1L 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 6.2).
• Seal vial

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 6Molarity (M) HCl (see guidance here)

  Safety information

  6M HCl should be handled under the chemical hood at all times. When storing under the chemical hood after use, be sure to place inside a secondary container to avoid spill accidents. The pH of HCl is 1.5 to 2.0

• Weigh 0.916g of La₂CO3 in weigh boat capable of holding 1mL of fluid

• Transfer the weigh boat and container to the chemical hood

• Pipette 0.1mL of HCl (6M) into the petri dish and carefully agitate the dish until all powder is dissolved

• Carefully transfer the solvated La₂CO₃solution 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 5.5), or within 2% of calculation based on pKa
• Seal vial

Prepare reaction vials

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

Add solution to reaction vial

• Pipette 3mL of 0.2Molarity (M) D-glucose solution to a glass reactor vial
• Add a nano stir bar to the reactor vial
• Add 3mL 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

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

  Note

  Critical step : When running for the first time, LACD1.1 will need to be created. Create a method that has a maximum set temperature of 170 °C, a time to ramp as 5 minutes with a hold time of 1 minute.

• 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= 0h 5m 0s

• Hold time= 0h 1m 0s

• 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).

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

Stabilize particles in buffer

• Prepare a sealed plastic or glass container that has 10mL of capacity and label (Lanthanum-doped carbon dots + KCl).

  Note

  Critical step : Be careful not to lose the nano stir bars, they are the most frequently misplaced item in this protocol.

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

  Note

  Note : If the recipe is altered, the storage buffer must be optimized. For this protocol, we used Zeta potential as an indicator of particle stability (analyzed daily over a 2 week experimental study)

• Inspect solution carefully for any colloids or particulate.

• Seal vial

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.

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).

  

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)

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.

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.