Biosensing in microfluidic channels using Fluorescence Polarization
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Biosensing in microfluidic channels using Fluorescence Polarization Vamsi K. Yadavalli and Michael V. Pishko Department of Chemical Engineering and the Materials Research Institute The Pennsylvania State University University Park, PA 16802, USA ABSTRACT Microfabricated microfluidic devices provide useful platforms for sensing and conducting immunoassays for high throughput screening and drug discovery. Here, Fluorescence Polarization has been used as a technique for probing binding events within 500 micron and smaller microfluidic channels fabricated in poly (dimethyl siloxane) (PDMS). The binding of concanavalin A to a lectin-dextran and a glycoproteinacetylcholinesterase has been used to demonstrate the homogeneous, ratioing format of fluorescence polarization for the quick and accurate determination of extremely low (nanomole) concentrations. Polarization has also been used to sense for a poly-aromatic hydrocarbon (PAH) within a microfluidic channel using binding to an antibody. We have also demonstrated a simple pH sensor based on the change in anisotropy of a pH sensitive fluorophore. The ease of fabrication and measurement using such polarization-based devices make them extremely suitable for micro-sized sensors, assays and total analysis systems. INTRODUCTION Fluorescence polarization is a technique that has been used to probe the orientation, mobility and interactions of molecules in biological and chemical systems [1, 2]. Optical sensing systems based on fluorescence polarization have also been studied to sense for analytes such as oxygen, glucose, pH and calcium [3-5]. These sensors have demonstrated the use of polarization as a simple method to create portable and accurate sensors. One of the biggest advantages of using fluorescence polarization as a technique in immunological probing and detection has been its simplicity and adaptability to miniaturization [6]. High speed and high throughput assays are possible because detection can usually be performed in a homogeneous format without the need for separating the free and bound probes [7]. Here, we describe the possibility of fluorescence polarization being used to probe for binding agents within 500 micron and smaller microfluidic channels containing mixing fluid streams. Protein binding has been demonstrated using a model lectin-glycoprotein and a lectin-sugar system. We have also demonstrated a polarization-based microsensor using the binding of a polyaromatic hydrocarbon molecule to a fluorophore-labeled antibody. The change in anisotropy of a pH dependent fluorophore upon exposure to microenvironmental pH changes has been further used to demonstrate a simple pH sensor within a channel. EXPERIMENTAL PROCEDURE
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Fluorescence spectra and anisotropy were recorded in an L-format configuration using a QM-2000 SE s
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