Development of a highly sensitive universal refractive index detector based on backscattering interferometry



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Texas Tech University


Refractive index (RI) detection is a common technique used in chemical and biochemical analyses. It can be employed to perform universal solute detection in micro-high performance liquid chromatography (ìHPLC), capillary electrophoresis (CE), as well as temperature measurements However, with current trends toward size reduction of fluid handling systems and the use of microfabricated devices in chemical and biochemical analyses accurate RI measurements in nanoliter of picoliter probe volumes present a significant challenge. A unique universal RI detector based or backscatter interferometry has been developed and has been shown to produce adequate performance for both capillary and "on-chip" detection. It consists of a coherent light source, microfluidic capillary or channel and a position sensitive phototransducer. When laser light strikes the microfluidic channel, a set of high contrast interference fringes appears in the direct backscattered region. The spatial distribution of theses fringes is directly proportional to the RI of the fluid contained within the channel. An alternative method for extracting RI information encoded in the backscattered fringes was developed based on spatial Fourier analysis. By monitoring phase in the Fourier domain detection limits of 7x10"^ RIU were achieved. Additionally, non-intrusive fluid flow measurements in small bore capillaries (with probe volume of 40 nL) and microfluidic channels (with probe volume of 300 pL) have been accomplished using a pump and probe configuration with backscatter interferometry. For the capillary 3-sigma detection limits were determined to be 0.71 nL/s, while for the microfluidic chips they were only 0.127 nL/s. In order to better understand how backscatter interferometry functions and to determine theoretical limits of the system a computer generated model was created using a sophisticated optical modeling program ASAP 6.5 (BRO Research, Inc). Models of capillary- and chip-based systems were created. The effects of such parameters as method of illumination, source wavelength, wall thickness, and channel dimensions were investigated. Furthermore, a completely different implementation of backscattered detector based on rectangular channels molded it poly(dimethyl)siloxane (PDMS) polymer has been developed. It was shown that Backscattering Interferometry in Rectangular Channels (BIRC) allows non-invasive label-free studies of protein - protein interactions within picoliter volumes. Quantification of irreversible streptavidin – biotin binding and reversible protein A - Human IgG Fc molecular interactions in a 225 picoliter detection volume was carried out. Detection limits of 47x10^-15 moles of biotin and 2x10^-15 moles of IgG - Fc were achieved.