Purpose
This protocol describes how to calibrate the P4SPR 2.0 using a series of NaCl solutions of known refractive index. Calibration establishes sensor sensitivity in RIU/nm and confirms the instrument is performing within specification.
Estimated Time
30 min
Sensitivity
≥ 1700 RIU/nm
R²
≥ 0.999
NaCl Solutions
Prepare five solutions of NaCl in distilled water. Prepare 10 mL of each. All solutions must be filtered (0.22 µm) and free of air bubbles before use.
| Concentration (mg/mL) | Refractive Index | Volume |
|---|---|---|
| 10 | 1.33429 | 10 mL |
| 20 | 1.33565 | 10 mL |
| 30 | 1.33699 | 10 mL |
| 40 | 1.33846 | 10 mL |
| 50 | 1.33999 | 10 mL |
Additional Materials
Instrument
- P4SPR 2.0, powered on and connected via USB-C
- Laptop running Windows 10 or 11, 64-bit
- AffiLabs.core installed and instrument detected
Consumables
- SPR sensor prism (unused or maintenance sensor)
- 1 mL syringes
- Filtered DI water (0.22 µm)
- Gloves
Setup
- Flat/low-profile waste container (5–7 cm from bench surface)
- Ambient temperature 15–37°C
- Humidity 20–70% RH
- Minimum 20 cm rear clearance
Protocol
Sensor Installation & Priming
- Wearing gloves, handle the sensor prism by its edges only. Install it into the instrument following the sensor installation procedure (User Guide, Chapter 9). Lower and lock the arm to press the microfluidic cell firmly against the sensor.
- Fill a 1 mL syringe with filtered DI water. Inject 500 µL into each channel inlet (A, B, C, D). Continue injecting until all air bubbles have cleared.
Warning: Never touch, clean, or wipe the gold surface. Air bubbles in the flow system will interfere with measurements.
Software Setup & Baseline
- Launch AffiLabs.core. The software automatically searches for the connected P4SPR 2.0 (30–45 s). When detected, the Calibration window appears. Complete the calibration step as prompted and proceed to Start Session.
- Allow 2 minutes for the DI water signal to stabilize. Monitor the sensorgram in the Live tab. Proceed only when drift is less than 0.5 nm/min.
- To switch the binding signal from RU to nm: go to Settings → Advanced Settings → nm.
Build the Calibration Method
- Open the Method Builder (Transport Bar → Build Method) and set up the following sequence:
| # | Cycle Type | Duration | Concentration | Notes |
|---|---|---|---|---|
| 1 | Baseline | 2 min | — | DI water equilibration |
| 2 | Binding | 3 min | 10 mg/mL NaCl | Lowest concentration |
| 3 | Binding | 3 min | 20 mg/mL NaCl | |
| 4 | Binding | 3 min | 30 mg/mL NaCl | |
| 5 | Binding | 3 min | 40 mg/mL NaCl | |
| 6 | Binding | 3 min | 50 mg/mL NaCl | Highest concentration |
| 7 | Baseline | 2 min | — | Post-run baseline check |
Tip: Always inject concentrations from lowest to highest (10 → 50 mg/mL) to minimize carryover between injections.
Recording & Injections
- Click Add to Queue, then click Start Cycle Record. Create a new folder for this experiment when prompted.
- During each Binding cycle, inject 200–300 µL of the corresponding NaCl solution into the sample channel at standard rate (~100 µL/s).
- Monitor the Manual Injection Assistant panel — the channel state changes from Ready → Binding → Wash automatically.
- When the Wash state is confirmed, proceed to the next Binding cycle. Repeat for all 5 concentrations.
- Allow the final Baseline cycle to complete, then click Stop (⏹). Final auto-save runs.
Data Processing & Sensitivity Calculation
Measure ΔSPR
- Navigate to the Edits tab. Click Load Data and select the auto-saved .xlsx file, or locate the experiment in the Notes tab and double-click to load.
- For each Binding cycle: place the green cursor at the start of injection and the red cursor at the end of the incubation period. The ΔSPR value updates live for all four channels.
- Keep the green cursor fixed at the start of injection. Move only the red cursor for the 20, 30, 40, and 50 mg/mL cycles. Record ΔSPR (nm) for each channel.
Tip: Measure all cycles at the same fixed timepoint after injection. Consistent endpoints are essential for a valid calibration curve.
Calculate Sensitivity
- In Excel, enter the refractive index for each concentration (from the table above) and the ΔSPR (nm) for each channel. Calculate the average ΔSPR across all channels.
- Plot RIU vs. average ΔSPR (nm). Fit a linear regression. The slope is the sensor sensitivity in RIU/nm.
- Record the R² value.
Acceptance Criteria
| Parameter | Acceptance Criterion |
|---|---|
| Sensor sensitivity | ≥ 1700 RIU/nm |
| R² (linear fit) | ≥ 0.999 |
| Baseline stability (pre-run) | Drift < 0.5 nm/min |
If sensitivity is below 1700 RIU/nm: Check for air bubbles in the channels, confirm the sensor is properly seated and the arm is locked, and repeat the run with a new sensor prism if necessary.
Shutdown
- Follow the shutdown and rinsing procedure (User Guide, Chapter 8).
- Remove sensor (User Guide, Chapter 9).
Related Protocols
- Protocol #1 — Covalent Immobilization of Proteins (EDC/NHS)
- Protocol #2 — Metal Chelation Immobilization of His-Tagged Proteins
- Protocol #3 — Streptavidin Capture of Proteins
PR-04 — Affinité Instruments Protocol Series · V4 (updated for AffiLabs.core) · April 2026