Applications of neutron reflectometry in biology

Over the last 10 years, neutron reflectometry (NR) has emerged as a powerful technique for the investigation of biologically relevant thin films. The great advantage of NR with respect to many other surface-sensitive techniques is its sub-nanometer resolution that enables structural characterizations at the molecular level. In the case of bio-relevant samples, NR is non-destructive and can be used to probe thin films at buried interfaces or enclosed in bulky sample environment equipment. Moreover, recent advances in biomolecular deutera-tion enabled new labeling strategies to highlight certain structural features and to resolve with better accuracy the location of chemically similar molecules within a thin film. In this chapter I will describe some applications of NR to bio-relevant samples and discuss some of the data analysis approaches available for biological thin films. In particular, examples on the structural characterization of biomembranes, protein films and protein-lipid interactions will be described.

Broadband Plasmon Waveguide Resonance Spectroscopy for Probing Biological Thin Films

A commercially available spectrometer has been modified to perform plasmon waveguide resonance (PWR) spectroscopy over a broad spectral bandwidth. When compared to surface plasmon resonance (SPR), PWR has the advantage of allowing measurements in both s- and p-polarizations on a waveguide surface that is silica or glass rather than a noble metal. Here the waveguide is a BK7 glass slide coated with silver and silica layers. The resonance wavelength is sensitive to the optical thickness of the medium adjacent to the silica layer. The sensitivity of this technique is characterized and compared with broadband SPR both experimentally and theoretically. The sensitivity of spectral PWR is comparable to that of spectral SPR for samples with refractive indices close to that of water. The hydrophilic surface of the waveguide allows supported lipid bilayers to be formed spontaneously by vesicle fusion; in contrast, the surface of an SPR chip requires chemical modification to create a supported lipid membrane. Broadband PWR spectroscopy should be a useful technique to study biointerfaces, including ligand binding to transmembrane receptors and adsorption of peripheral proteins on ligand-bearing membranes.

Fixed polarizer ellipsometry for simple and sensitive detection of thin films generated by specific molecular interactions: applications in immunoassays and DNA sequence detection

Biological thin films may form on a surface by specific molecular interactions. The fixed polarizer ellipsometer (FPE) is a sensitive instrument that detects biological thin films either qualitatively or quantitatively. The design is simple and inexpensive. The assays are formatted on an optical surface, and the FPE detection is based on the phase shift of linearly polarized light after reflection through a thin film. We have constructed mathematical models of the FPE response to reflection through single-layer and two-layer films that agree closely with experimental data. Several biological assays have been measured with the FPE to demonstrate the application of this technology to clinical targets, including ultrasensitive immunoassays for hepatitis B surface antigen (0.1 ng/mL) and α-fetoprotein (0.01 ng/mL) and DNA hybridization (0.5 fmol/μL target probe). A clinical study for detection of group A streptococcus from patient throat swabs demonstrated the qualitative application of the FPE to infectious disease targets. The flexibility and sensitivity of the FPE makes this technology suitable for numerous target analytes and applications.