Surface second harmonic generation (SHG) is a coherent, nonlinear optical technique that is well suited for investigations of biomolecular interactions at interfaces. SHG is surface specific due to the intrinsic symmetry constraints on the nonlinear process, providing a distinct analytical advantage over linear spectroscopic methods, such as fluorescence and UV-Visible absorbance spectroscopies. SHG has the ability to detect low concentrations of analytes, such as proteins, peptides, and small molecules, due to its high sensitivity, and the second harmonic response can be enhanced through the use of target molecules that are resonant with the incident (ω) and/or second harmonic (2ω) frequencies. This review describes the theoretical background of SHG, and then it discusses its sensitivity, limit of detection, and the implementation of the method. It also encompasses the applications of surface SHG directed at the study of protein-surface, small-molecule-surface, and nanoparticle-membrane interactions, as well as molecular chirality, imaging, and immunoassays. The versatility, high sensitivity, and surface specificity of SHG show great potential for developments in biosensors and bioassays.

A comparison of the binding properties of avidin, streptavidin, neutrAvidin, and antibiotin antibody to a biotinylated lipid bilayer was studied using second-harmonic generation. Protein binding assays were performed on a planar supported lipid bilayer of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) containing 4 mol % biotinylated-cap-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (biotin-cap-DOPE). The equilibrium binding affinities of these biotin-protein interactions were determined, revealing the relative energetic contributions for each protein to the biotinylated lipid ligand. The results show that the binding affinities of avidin, streptavidin, and neutrAvidin for biotin were all strengthened by protein-protein interactions but that the stronger protein-protein interactions observed for streptavidin and neutrAvidin make their binding more energetically favorable. It was also shown that neutrAvidin has the highest degree of nonspecific adsorption to a pure DOPC bilayer, compared to avidin and streptavidin. In addition, the biotin-binding affinity of the antibiotin antibody was found to be of the same order of magnitude as that of avidin, streptavidin, and neutrAvidin. These findings provide important new insights into these biotin-bound protein complexes commonly used in several bioanalytical applications.

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.

Binding kinetics of the multivalent proteins peanut agglutinin (PnA) and cholera toxin B subunit (CTB) to a GM1-doped 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid bilayer were investigated by both second-harmonic correlation spectroscopy (SHCS) and a traditional equilibrium binding isotherm. Adsorption and desorption rates, as well as binding affinity and binding free energy, for three bulk protein concentrations were determined by SHCS. For PnA binding to GM1, the measured adsorption rate decreased with increasing bulk PnA concentration from (3.7 ± 0.3) × 106 M–1·s–1 at 0.43 μM PnA to (1.1 ± 0.1) × 105 M–1·s–1 at 12 μM PnA. CTB–GM1 exhibited a similar trend, decreasing from (1.0 ± 0.1) × 109 M–1·s–1 at 0.5 nM CTB to (3.5 ± 0.2) × 106 M–1·s–1 at 240 nM CTB. The measured desorption rates in both studies did not exhibit any dependence on initial protein concentration. As such, 0.43 μM PnA and 0.5 nM CTB had the strongest measured binding affinities, (3.7 ± 0.8) × 109 M–1 and (2.8 ± 0.5) × 1013 M–1, respectively. Analysis of the binding isotherm data suggests there is electrostatic repulsion between protein molecules when PnA binds GM1, while CTB–GM1 demonstrates positive ligand–ligand cooperativity. This study provides additional insight into the complex interactions between multivalent proteins and their ligands and showcases SHCS for examining these complex yet technologically important protein–ligand complexes used in biosensors, immunoassays, and other biomedical diagnostics.

Lens-less surface second harmonic generation imaging (SSHGI) is used to image an SHG active molecule, (S)-(+)-1,1'-bi-2-naphthol (SBN), incorporated into a lipid bilayer patterned with the 1951 United States Air Force resolution test target. Data show the coherent plane-wave nature of SHG allows direct imaging without the aid of a lens system. Lens-less SSHGI readily resolves line-widths as small as 223 μm at an object-image distance of 7.6 cm and line-widths of 397 μm at distances as far as 30 cm. Lens-less SSHGI simplifies the detection method, raises photon collection efficiency, and expands the field-of-view. These advantages allow greater throughput and make lens-less SSHGI a potentially valuable detection method for biosensors and medical diagnostics.