Second Harmonic Correlation Spectroscopy: A Method for Determining Surface Binding Kinetics and Thermodynamics

Abstract

These studies describe the implementation of second harmonic correlation spectroscopy (SHCS) to measure the adsorption and desorption kinetics of molecular species associated with a surface. Specifically, the local fluctuations of the measured second harmonic (SH) signal were used to determine the binding kinetics and thermodynamics of (S)-(+)-1,1'-bi-2-napthol SBN intercalation into a 1,2-dioleoyl-sn-glycero-3-phosphocoline (DOPC) bilayer. In order to determine the adsorption and desorption rates, the SH signal was collected above saturation concentration at steady-state equilibrium as a function of time. The autocorrelated SH signal was then fit to a correlation model developed for molecules binding at a surface when there is no contribution from molecules in solution. The measured adsorption rate for SBN to DOPC was 2.7 ± 0.2 × 10(3) s(-1) M(-1) and the desorption rate was 9 ± 4 × 10(-4) s(-1). The kinetic rates as well as the calculated equilibrium binding constant, 3.0 ± 1.3 × 10(6) M(-1) obtained from SHCS were compared with those obtained from a conventional binding isotherm and found to be statistically consistent. The primary advantage of using SHCS is both the absorption and desorption rates were determined in the same experiment using only a single bulk concentration of SBN. The results of these studies demonstrate that SHCS can be used to provide accurate kinetic and thermodynamic binding data in a label-free manner in lieu of conventional isotherm studies, especially where time and analyte are scarce.

Determination of Multivalent Protein–Ligand Binding Kinetics by Second-Harmonic Correlation Spectroscopy

“… 29 In the same study, correlation data of a pure DOPC bilayer without addition of any SBN was also investigated and displayed no correlated events, further demonstrating the absence of correlated proportional noise or correlated noise from the bilayer. 29 …”

“…30 − 32 The surface specificity of SHCS eliminates the contributions from diffusion of molecules in solution, such that the only contributing factors to the correlated fluctuations in the SH signal are from the surface binding kinetics. 29 …”

“… 26 − 28 More recently, SHCS was used to accurately determine the binding kinetics of the small molecule ( s )-(+)-1,1′-bi-2-naphthol (SBN) intercalating into a PSLB. 29 The current study is the first to extend the SHCS technique to detection and investigation of protein binding at a surface. Use of SHCS to measure the binding kinetics separately for several bulk protein concentrations, as well as to examine the cooperative and electrostatic contributions of these multivalent protein–ligand interactions, provides additional insight for their use in biosensors, medical diagnostics, and drug development.…”

Second Harmonic Correlation Spectroscopy: Theory and Principles for Determining Surface Binding Kinetics

“…17, both the adsorption and desorption rate can be retrieved from the measured fluctuations in SH intensity for a single analyte concentration; however, to improve the error of the fitting parameters (N s , k on , and k off ) a separate measurement of the desorption rate can be beneficial, but not wholly necessary. 20,21 When the mechanism of binding does not change with concentration, collecting correlation data from multiple bulk concentrations can also provide the adsorption and desorption rates with increased confidence and reduced error. Monovalent binding, such as SBN intercalating into a DOPC bilayer, generally does not show concentration dependent binding kinetics and is an excellent system to globally fit correlation data to determine both the adsorption and desorption rates of binding.…”

“…In particular, second harmonic correlation spectroscopy (SHCS) has been used to determine the diffusion coefficient of dye aggregates 17 and long chain para substituted aromatic amphiphiles at a surface. 19 The authors have previously demonstrated the ability of SHCS to determine the binding kinetics of a monovalent interaction between the small molecule ( s )–(+)–1,1’-bi-2-naphthol (SBN) and a 1,2-dioleoyl- sn -glycero-3-phosphocoline (DOPC) lipid bilayer 20 and the more complex interaction between the multivalent proteins cholera toxin b subunit (CTb) and peanut agglutinin (PnA) with monosialotetrahexosylganglioside (GM 1 ) doped DOPC bilayers. 21 These previous studies also illustrate the shortcomings of obtaining surface binding kinetics through traditional adsorption isotherms combined with desorption experiments.…”