Chapter Four · failure evidence
What Receptor Binding Kinetics & SPR got wrong, from 37 dissertations
The records evaluate various experimental techniques and analytical models applied to receptor binding kinetics, surface plasmon resonance, and associated biophysical assays. Across these studies, researchers encountered repeated challenges involving surface fouling, detrimental immobilization chemistry, kinetic fitting failures, and analyte properties that violated assay assumptions. These records come from PhD theses at 16 institutions, 2021 to 2026. Each links to its thesis. They were extracted by language models reading the full text, so treat each as a lead to read, not a verdict.
Non-specific adsorption and surface fouling obscure specific interaction signals
Investigators found that non-specific background adsorption and irreversible protein deposition frequently masked target-specific binding signals across biosensor surfaces. These fouling phenomena created thick surface layers, caused false-positive screening results, and prevented the observation of saturable high-affinity binding sites.
Tried and failed
surface plasmon resonance and quartz crystal microbalance applied to protein binding and unbinding kinetics. Reason: severe protein aggregation and irreversible non-specific deposition formed thick layers on the sensor surface
Biophysical Dynamics Of Rgs-Lov Proteins As Systems For Light-Induced Membrane Recruitment · Penn
Tried and failed
affinity enrichment without signal amplification applied to secreted glycoproteins in serum-containing media. Outcome: no signal. Reason: Non-specific binding of high-abundance background proteins overwhelmed target glycoprotein detection.
MS-Based Chemical Proteomics Studies of Extracellular Glycoproteins: Identification, Quantification, and Dynamics · Georgia Tech
Tried and failed
SPME-based measurement of specific protein binding affinity applied to long-chain hydrophobic surfactant-protein interactions. Outcome: no signal. Reason: Non-specific sorption dominated, masking any saturable high-affinity specific binding sites on the protein
Toxic Effects and Exposure of Per- and Polyfluoroalkyl Substances in Cell-based Bioassays · Publikationssystem UB Tuebingen
Considered and rejected
Considered and rejected: Rejected poly-L-lysine (PLL) slide coating due to prominent non-specific probe binding background
Single-cell imaging assay for rapid antimicrobial resistance testing · Oxford
Considered and rejected
Considered and rejected: Ceased testing synthetic peptide binders (RRGW, WGRR, Fc-III-4C) because binding signal was below or equivalent to non-specific background binding to ELISA plates.
Collagen Anchoring Agonist Antibodies for Cancer Immunotherapy · MIT
Tried and failed
screening cyclic peptide libraries at low ionic strength applied to protein-protein interaction inhibitor discovery. Reason: low ionic strength causes non-specific electrostatic binding and high false-positive rates
Discovery of macrocyclic inhibitors of challenging protein-protein interactions · EPFL
Considered and rejected
Considered and rejected: Rejected direct physical adsorption antibody immobilization due to excessive non-specific binding and non-uniform patchiness across gold wires.
Microfluidics Sensor to Detect Water Salinity and Bacteria Using Optical and Electrical Methods · YorkSpace
Suboptimal immobilization chemistry and regeneration degrade sensor surface activity
Standard covalent coupling protocols and harsh regeneration conditions frequently disrupted ligand binding competence or stripped active complexes from sensor chips. Furthermore, excessive surface ligand packing density and long linkers induced severe steric hindrance that blocked analyte access.
Tried and failed
amine coupling surface plasmon resonance immobilisation applied to recombinant protein on sensor chip. Reason: protein failed to immobilise under standard low pH sodium acetate buffer conditions
Tried and failed
prolonged covalent cross-linker incubation time applied to impedimetric biosensor surface functionalization. Outcome: worse than baseline. Reason: surface saturation and steric hindrance reduced subsequent antibody binding and signal response
Development of an impedimetric biosensor for lung cancer detection. · Cranfield
Tried and failed
high surface density ligand immobilization applied to biosensor nanoparticle binding detection. Outcome: worse than baseline. Reason: excessive surface packing density caused severe steric hindrance that impeded analyte binding kinetics and affinity
Tried and failed
immobilizing empty protein heterodimers on sensor chips applied to ligand binding kinetics measurement. Outcome: unstable. Reason: complexes dissociated rapidly upon immobilization without a stabilizing ligand present
A Complete Pipeline for the Discovery and Development of Haptenizing Chimeras at Scale · Harvard
Tried and failed
acidic surfactant regeneration of immobilized antibody sensor applied to surface plasmon resonance biosensor chips. Outcome: unstable. Reason: harsh regeneration conditions degraded immobilized antibody binding capacity after few cycles
Biological and artificial receptors in affinity sensor for water toxins detection · Cranfield
Considered and rejected
Considered and rejected: Rejected amine coupling of galectins directly to SPR chips because covalent immobilization disrupted oligomerization and caused loss of binding activity.
Defining the mechanism of galectin-3-mediated TGF-β1 activation and its role in lung fibrosis · University of Nottingham Repository
Considered and rejected
Considered and rejected: Rejected long-linker biotinylation reagents (20,000 g/mol) despite superior MACS capture efficiency due to steric hindrance attenuating scFv-antigen binding.
Developing an antibody discovery platform for targeting membrane proteins via cell-cell screening · JScholarship
Complex reaction pathways and rapid rates prevent reliable kinetic fitting
Kinetic fitting routines broke down when association and dissociation rates were too rapid for instrument temporal resolution or when multi-site binding prevented saturation. Unstirred-layer diffusion artifacts and concentration anomalies similarly produced distorted sensorgrams and led to the rejection of proposed kinetic models.
Tried and failed
surface plasmon resonance binding affinity modeling applied to fragment library screening on structured RNA. Outcome: did not converge. Reason: complex multi-site binding and solubility limits prevented reaching surface saturation needed for kinetic fitting
Tried and failed
sequential multi-site binding model applied to isothermal titration calorimetry data. Outcome: did not converge. Reason: The model failed to adequately fit the experimental titration curve.
C2 domains of dysferlin and synaptotagmin-1: Structure and mechanics · Texas Tech
Tried and failed
surface plasmon resonance kinetic fitting applied to fast-exchanging protein-protein interactions. Reason: association and dissociation rates were too fast to be resolved within instrument temporal limits
CHARACTERIZING THE STRUCTURE AND DYNAMICS OF ADAPTIVE IMMUNE PROTEINS TO INFORM IMMUNOTHERAPY DESIGN · Penn
Considered and rejected
Considered and rejected: Rejected pure GUV transfer assay for studying association kinetics of BAR proteins at <200 nM due to severe unstirred-layer diffusion artifacts producing sigmoidal curves.
Mechanisms of Membrane Remodeling by Peripheral Proteins and Divalent Cations · Penn
Considered and rejected
Considered and rejected: Rejected Sequential Binding-Dimerization Model because the observed second rate increased with lipid concentration and showed inverse/no dependence on peptide concentration, which contradicts membrane surface dilution kinetics.
Spectroscopic Studies of Peptide-Membrane Interactions · Penn
Considered and rejected
Considered and rejected: Discontinued SPR screening for small-molecule KD determination after recurring sensorgram distortions and non-saturating kinetics.
The development and evaluation of PET imaging agents targeting PD-L1 · Imperial
Analyte heterogeneity and oligomerization violate standard biosensor assay requirements
Surface plasmon resonance and related binding assays yielded unreliable data when measuring large, heterogeneous complexes or analytes prone to oligomerization at required assay concentrations. In other instances, standard in vitro sensor chips induced irreversible binding artifacts that failed to replicate native physiological behavior.
Considered and rejected
Considered and rejected: Rejected using isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) for the benzophenanthridinone class (968) due to high protein concentration requirements where GAC forms dimers/tetramers rather than binding monomers.
THE ALLOSTERIC REGULATION OF GLUTAMINASE ENZYMES: DRIVING GLUTAMINE CATABOLISM IN CANCER CELLS · Cornell
Considered and rejected
Considered and rejected: Standard binding assays (QCM, SPR, floatation assays, fluorimetry, calorimetry) were rejected in favor of high-throughput AR-SHS because QCM loses sensitivity below nanogram protein weights, SPR requires kinetic modeling limiting sensitivity to the micromolar range, fluorimetry requires labeling that perturbs native binding affinity, and calorimetry lacks binding-event specificity.
Second harmonic scattering of water in a biological context · EPFL
Considered and rejected
Considered and rejected: Rejected surface plasmon resonance (SPR) for determining LO1 and LO1Fab binding kinetics because the large size and heterogeneity of MDA-LDL yielded unreliable and inaccurate data; adopted microfluidic diffusional sizing instead.
Considered and rejected
Considered and rejected: Rejected using permanently locked mutants (C292A, Q355N) and in vitro SPR data for kinetic parameters due to irreversible SLB binding and lack of cellular milieu fidelity.
Biophysical Dynamics Of Rgs-Lov Proteins As Systems For Light-Induced Membrane Recruitment · Penn
Low refractive index contrast and optical artifacts prevent detectable sensor responses
Optical transducers frequently failed to register binding events due to insufficient mass density or inadequate refractive index contrast between bound analytes and the sensor surface. Additionally, severe light scattering artifacts and omitted capture ligands prevented the generation of concentration-dependent optical shifts.
Tried and failed
responsive polymer shell coating for plasmonic biosensing applied to protein detection via LSPR shifts. Outcome: no signal. Reason: Target protein binding caused no concentration-dependent localized surface plasmon resonance wavelength shifts across various polymerizations
Tried and failed
plasmonic biosensing without specific capture ligands applied to single-cell secretion detection. Outcome: no signal. Reason: lack of affinity capture ligands prevents target analyte accumulation on the sensor surface
Tried and failed
direct protein physisorption onto sensor surface applied to optical biosensing transducers. Outcome: no signal. Reason: insufficient surface binding density or low refractive index contrast prevents measurable optical resonance shifts
All-Dielectric Nanophotonic via Glass Fluid Instabilities · EPFL
Tried and failed
fluorescence anisotropy binding assay applied to protein-lipid vesicle interactions. Outcome: no signal. Reason: excessive light scattering artifacts at required millimolar lipid concentrations overwhelmed the fluorescence signal
Left open by the authors
Problems the authors named and did not get to.
Left open
Assess antibody affinity and somatically hypermutated clones using surface plasmon resonance and B-cell sequencing instead of ELISA titers. Blocker: Requires a wet lab, animal/patient samples, surface plasmon resonance apparatus, and B-cell sequencing facilities.
Mesoporous Silica Rods Scaffolds for the generation of adaptive immune responses · Harvard
Left open
Identify additional hits from antibody libraries and characterize full IgG binding affinity using surface plasmon resonance. Blocker: Requires wet lab facilities, surface plasmon resonance equipment, and physical antibody libraries/samples
ENGINEERING THE HUMORAL RESPONSE TO GENERATE ANTIGEN-SPECIFIC ANTIBODIES · Cornell
Left open
Characterize neurolysin activator-induced conformational shifts using fluorescence polarization, X-ray crystallography, surface plasmon resonance, or molecular dynamics simulations. Blocker: Requires wet-lab experimental apparatus (X-ray crystallography, SPR, FP assays) and physical protein/compounds for experimental validation.
Discovery of small molecule allosteric modulators for endopeptidase neurolysin · Texas Tech
Left open
Validate binding affinity of ambiguous or fluorescence-quenching MST hit compounds against G9a using surface plasmon resonance assays. Blocker: Requires a wet lab, surface plasmon resonance instrumentation, purified recombinant G9a protein, and physical compound samples
Left open
Validate and implement synthetic NES peptides instead of MBP-NES fusion proteins in high-throughput CRM1 fluorescence polarization binding assays. Blocker: Requires a biochemistry wet lab, physical peptide synthesis, purified CRM1 protein, and fluorescence polarization assay equipment.
Nuclear Export Signal Recognition by CRM1 Carrying the Oncogenic E571K Mutation and Structure-Based NES Prediction · DSpace at UTSWMED
Left open
Optimize and repeat surface plasmon resonance (SPR) binding kinetics measurements between knuckle peptides and BMP receptors. Blocker: Requires wet lab access, an SPR instrument, synthesized knuckle peptides, and purified BMP receptors
Left open
Perform SPR, SERS, or QCM measurements to validate binding affinity between the synthesized nanoMIPs and target CD45 protein. Blocker: Requires wet lab facilities, analytical instruments (SPR/SERS/QCM), and physical nanoMIP and protein samples.
Left open
Perform direct binding validation and biophysical characterization of non-aromatic bacterial quorum sensing molecule DPD with AhR. Blocker: Requires wet lab assays and biophysical characterization apparatus (e.g., purified AhR protein and DPD binding assays)
Left open
Test ribosome binding sites of varying strengths to measure the effect of different translation rates on plasmid crosstalk in cell-free biosensing. Blocker: Requires molecular biology wet lab experiments with plasmid variants in cell-free extracts.
Towards Robust, Point-of-Care Biosensing in Cell-Free Systems · Georgia Tech
Left open
Test whether a simple cell or protein binding assay predicts loss of binding and in vivo transcytosis activity of antibody mutants. Blocker: Requires wet lab experiments with cell or protein binding assays and mutant antibodies
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