Neurotransmitter Diagnostics Platform

1. Chitosan Sol-Gel Solution Preparation

A key element of the biochip is the selective sensing membrane. Chitosan polymer is dissolved to create a robust, hydrophilic network supporting neurotransmitter electron transfer:

1. Weighing: Weigh exactly 100 mg of high-deacetylation chitosan powder (minimum 90% degree of deacetylation).
2. Acid dissolution: Measure 10 mL of distilled water and add 100 µL of glacial acetic acid to create a 1.0% (v/v) aqueous acetic acid solvent.
3. Mixing: Slowly disperse the chitosan powder into the acidic solution. Stir continuously at 50°C for 2 to 3 hours until a completely clear, transparent, viscous sol-gel matrix is formed.
4. pH Adjustment: Adjust the pH of the gel to approximately 4.5 - 5.0 using a dilute sodium hydroxide (NaOH) solution. This maintains polymer stability and supports electrochemical enzyme/mediator activities.
5. Storage: Store the prepared sol-gel at 4°C. The matrix is stable for up to 3 weeks.

2. SPE Surface Modification Protocol (Primary Build)

The primary working electrode is the Screen Printed Carbon Electrode (SPE) with Adapter. Unlike GCE, the SPE does not require mechanical polishing — it is a disposable single-use carbon strip. Modification steps:

• Step A (Surface Cleaning): Rinse the SPE carbon working area gently with distilled water and air-dry for 5 minutes. Do not mechanically polish — the screen-printed surface is not designed for abrasive treatment.
• Step B (Nanocomposite dispersion): Mix 1.0 mg/mL of Graphene Oxide (GO) into 1 mL of the chitosan sol-gel. Sonicate for 20 minutes to achieve a uniform carbon nanocomposite suspension. CNTs may be substituted if GO is unavailable.
• Step C (Drop Casting): Drop-cast exactly 5 µL to 8 µL of the Chitosan/GO nanocomposite gel onto the SPE carbon working electrode area using a micropipette.
• Step D (Evaporation / Drying): Place the modified SPE under an inverted beaker at room temperature for 12 hours. This forms a thin, ion-conductive, biocompatible sensing interface.

GCE (Backup): If using GCE for comparison, polish with alumina slurry (0.3 µm then 0.05 µm) on microcloth, rinse, sonicate in ethanol 3 min, then distilled water 3 min, and air-dry before drop-casting.

1. Cyclic Voltammetry (CV) Sweep

Cyclic Voltammetry is used to identify the characteristic oxidation-reduction (redox) voltage peaks of the target neurotransmitters:

• Fill the electrochemical cell cup with 10 mL of 0.01M PBS buffer solution (pH 7.4).
• Immerse the 3 electrodes (GCE modified with Chitosan, Ag/AgCl, and Graphite stick) into the buffer.
• In the dashboard, trigger a **CV Sweep** from **-0.2V to +0.8V** at a scan rate of 50 mV/s.
• This initial scan serves as the baseline measurement. The current curve should look smooth without oxidation peaks.

2. Neurotransmitter Calibration Steps

To measure concentrations and verify sensitivity:

• Dopamine detection: Inject 100 µL of 1.0 mM Dopamine solution into the PBS cell. Run a CV Sweep. You will observe a distinct oxidation peak around **+0.22 V** representing Dopamine conversion to Dopaminequinone.
• Serotonin detection: Run a scan in a cell containing Serotonin. You will observe a distinct oxidation peak shifted further anodic around **+0.38 V**.
• Amperometric read: Under continuous monitoring, hold the potential fixed at **+0.22 V**. When dopamine is added, the current will spike in real-time, showing a proportional relationship with dopamine concentration.