Focal molography with internal nanopattern referencing delivers accurate affinity, kinetic, and concentration measurements in cell culture media and serum. In these matrices, SPR and BLI struggle with non-specific binding and baseline drift.

The Challenge

Affinity, kinetic, and concentration measurements in complex matrices such as serum, cell culture media, and supernatants are central to drug discovery, biomarker research, and bioprocessing. Yet gold-standard surface methods like SPR and BLI suffer from non-specific binding and baseline drift. They also require precisely analyte-depleted matched serum, samples that are often not available, affordable, or well matched in practice.

The Approach

The authors benchmarked focal molography head-to-head against SPR (Biacore) and BLI (Octet) for K_D, k_on, k_off, and direct protein quantification. Four matrices were compared: PBST, PBST+C with blocking proteins, TexMACS cell culture medium, and 50% bovine serum. Three model systems were tested on the MACS® Matchmaker with DNA-directed immobilization (DDI) of single-domain heavy-chain antibodies. They were αCD4/sCD4, αGFP/GFP, and the challenging sticky target Granzyme B.

Key Results

• Cross-method agreement: K_Ds were within 1.9-fold of each other across focal molography, BLI, and SPR for αCD4/sCD4 in buffer with blocking proteins. In TexMACS cell culture medium they were within 2.4-fold. This confirms focal molography as quantitatively equivalent to SPR and BLI
• Stable measurements in 50% bovine serum: Focal molography kept stable baselines. It yielded K_Ds for sCD4 within 1.8-fold of standard-buffer values (48 nM vs 27 nM) without external referencing. SPR and BLI required analyte-depleted matched serum, and SPR even showed sign inversion during analyte injection
• Robustness to sticky non-specific binders: Granzyme B is a sticky target. Focal molography determined K_D (18.3 nM), k_on, and k_off directly in cell culture medium using only internal referencing. SPR and BLI produced kinetic constants outside instrument specifications even after extensive biochemical matching and double referencing
• Direct quantification: sCD4 spiked at 50 nM was recovered at 97.8 to 100.3% accuracy in TexMACS and 99.0% in 50% FBS. The inter-assay CV was 1.2% across three experiments, and the LoD 2.6 nM in serum
• On-chip multiplexing capability: Using just 4 of 54 molograms per analyte still delivered intra-assay CVs below 10% and recoveries within ±10%. That allows simultaneous quantification of up to 13 analytes on a single chip