Binding Characterization: SPR and BLI

SPR vs BLI Decision Matrix

Factor	Choose SPR	Choose BLI
Sensitivity	Small molecules, fragments (<500 Da)	Large complexes, antibodies
Throughput	Low-medium (serial)	High (96-well parallel)
Sample purity	Required (clogs fluidics)	Tolerates crude lysates
Kinetic resolution	Higher (better for fast kinetics)	Lower
Mass transport	More sensitive (may distort kon)	Less sensitive
Maintenance	High (fluidics system)	Low (dip-and-read)
Sample consumption	Higher (continuous flow)	Lower
Cost per experiment	Lower chip cost, higher run cost	Higher tip cost, lower run cost

Key differences

SPR (Surface Plasmon Resonance)

• Mechanism: Detects refractive index changes at gold surface
• Surface: Gold chip with dextran matrix (CM5, CM7, etc.)
• Flow: Continuous microfluidics
• Best for: Small molecules, high-affinity, precise kon/koff

BLI (Biolayer Interferometry)

• Mechanism: Measures optical interference pattern shift
• Surface: Fiber optic biosensor tips (SA, Ni-NTA, AHC)
• Flow: Dip-and-read (no microfluidics)
• Best for: High-throughput, crude samples, antibody screening

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Troubleshooting: Why BLI works but SPR doesn't

Cause	Mechanism	Solution
Hydrophobic CDRs	Adsorb to SPR gold/dextran surface	Add 0.05% Tween-20, use CM7 chip with longer dextran
Aggregation	Mass transport artifacts in SPR fluidics	Filter sample (0.22μm), reduce ligand density
High instability	Degrades during continuous flow	Shorter cycle time, add stabilizers (trehalose 5%)
Charge mismatch	Nonspecific binding to charged dextran	Adjust buffer pH ±1 from pI, add BSA 1mg/mL
Slow dissociation	Long regeneration needed (damages ligand)	Use BLI (disposable tips)