TITLE: Fabrication of dip-and-read orthophosphate chemosensor based on laser inscribed graphene
AUTHORS: Geisianny A Moreira, Alex Shaw, Eric S McLamore

[DESCRIPTION]
This protocol describes the fabrication of a LIG dip-and-read orthophosphate sensor based on a 2 dimensional sorbent structure (poly[diallydimethylammonium] chloride, a.k.a, PolyDADAMAC) grafted on graphene oxide (GO) as recognition element. The ortho-P sensor uses electrochemical impedance spectroscopy (EIS) as a transduction technique, and laser-induced graphene electrodes as a transducer platform. The process requires approximately 5 hours (from sensor fabrication to testing)



 



[BEFORE_START]
Other protocols required
LIG fabrication (Link)
LIG 3-electrode plug-and-play chip (Link)
Cyclic voltammetry using ferrocyanide/ferricyanide couple (Link)

[GUIDELINES]
Be sure to wear appropriate safety PPE throughout (lab coat, gloves, eyewear).
Electronic or physical lab notebook may be used throughout

[STEPS]
SECTION: Step 1) Prepare working solutions
1. Prepare Solution 1: 1 mg/mL GO-PDDA solution
Prepare a working solution for GO-PDDA at 1 mg/mL by diluting the stock solution (5 mg/mL) in DI water.
Agitate using a vortex mixer until the solution is dispersed 
SECTION: Step 1) Prepare working solutions
2. Prepare Solution 2: 2X Sodium chloride/sodium bicarbonate (NaCl/NaHCO3) solution
Add two packets of buffer (isotonic buffer pouches) to 240 mL of DI water.
Agitate with vortex mixer until dissolved
SECTION: Step 1) Prepare working solutions
3. Prepare Solution 3: 100 mg/L phosphate, nitrate, sulfate, chloride salt solutions
Dilute standard solutions of phosphate, nitrate, sulfate and chloride (1000 mg/L) in DI water to get a final concentration of 100 mg/L for each ion separately. 
Working solutions may be prepared in 1.5 mL microcentrifuge tubes if desired
SECTION: Step 2) Functionalize LIG electrodes
4. Follow LIG fabrication protocol (Link)
Next, prepare 3-electrode plug-and-play chip using the published protocol (Link)
After fabricating LIG electrodes, follow CV protocol (Link) 
Follow the quality control (QC) guideline in Qian et al (Link) to select triplicates 

 

Pipette 10 mL of GO-PDDA working solution (1 mg/mL) onto the LIG working electrode. Be careful that the solution does not coat reference and/or counter electrodes. 
Allow the GO-PDDA working solution to dry at room temperature for at least 20 minutes. 
Repeat these steps two more times; pipette 10 mL of GO-PDDA solution and then dry at room temperature. 

 

After LIG functionalization, run an additional CV test in ferro/ferricyanide solution (Link) to confirm surface coating is stable. See Figure 1D for expected results
If available, use a stereo microscope to image the surface and inspect for homogenous coating 

 

SECTION: Step 3) Characterize sensor performance
5. Add 15 mL of 2X Sodium chloride/sodium bicarbonate (NaCl/NaHCO3) solution to an electrochemical cell.
Immerse LIG-GO-PDDA sensor into the solution.

 

Conduct Run EIS for baseline measurements following the settings below:

 

Record the raw data of baseline measurements for each replicate in an Excel file (Template).
Titrate stock solution (e.g., 100 mg/L of phosphate, nitrate, sulfate or chloride salt). An example calibration experiment is shown in the Table below



 

After analyte injection, mix the buffer solution with a pipette, allow the solution to sit for one minute, and then run EIS measurements for each concentration.
Record the raw data of different analyte concentrations for each replicate in an Excel file (Template).
SECTION: Step 4) Clean up space and dispose of waste
6. Dispose of used chemicals according to the lab safety plan.
Wash all shot glass cups with mild detergent and warm water.