Affinity Detection of Low Molecular Weight Analytes

Abstract

In this paper we report attempts to detect directly the binding of a low molecular weight substance to a protein binding site. An optical transducer based on reflectometric interference spectroscopy (RIFS) was used to detect the binding of biotin (244 g/mol) to a thin silica film surface coated with streptavidin. RIFS allows measurement of changes in the optical thickness of thin transparent films with high resolution. During immobilization of streptavidin, an increase in layer thickness of about 5 nm was detected. Subsequent incubation with biotin (4 μM) resulted in a thickness increase of about 70 pm. Repeated incubation with biotin gave no further increase in layer thickness. The lowest biotin concentration showing significant effects was 40 nM. Incubation with benzoic acid (40 μM) gave no thickness change. The setup allowed significant detection of thickness increases of 2 pm and above. Therefore, the thickness effects observed in the study could be unambiguously and clearly identified.

Label-free and dynamic detection of biomolecular interactions for high-throughput microarray applications

“…S1. Thus, we choose to interpret our OPD signal due to biomolecule binding as a change in thickness at a constant refractive index of 1.45, commonly used as the refractive index of a monolayer of protein (15,16). This does not necessarily yield the actual thickness because the refractive index of the molecular layer may be different, but it does yield the actual surface-adsorbed mass density calculated from the measured thickness.…”

“…This does not necessarily yield the actual thickness because the refractive index of the molecular layer may be different, but it does yield the actual surface-adsorbed mass density calculated from the measured thickness. It was shown by radiolabeling methods that in similar optical detection methods, 1 pm height change corresponds to Ϸ1 pg/mm 2 of binding material on the surface (15) or to a Ϸ1 ϫ 10 Ϫ5 change in refractive index (17).…”

“…Picometer noise floor was shown by several researchers (15,18), but, when imaging a multiplexed array is required, the reported sensitivity drops. Averaging improves sensitivity by increasing the total number of detected photons and can reduce the effects of surface roughness.…”

Effective-substrate theory for optical reflection from a layered substrate

“…(11) and Eq. (12), we find (13) with (14) with and are given by Eq. (10) , such that the reflection from such a system is given by (15) (16) Here is found iteratively using the following equations,…”

“…Optical reflection techniques such as ellipsometry [1][2][3][4][5][6], surface plasmon polariton wave excitation [7][8][9][10][11], spectral interferometry [12][13][14], reflection anisotropy spectroscopy [15,16] and surface photo-absorption [17], are widely used to monitor processes at surfaces and interfaces of solids. They are desirable for versatility, non-invasiveness, and ease of implementation.…”