A plastic colorimetric resonant optical biosensor for multiparallel detection of label-free biochemical interactions

Abstract

A high sensitivity plastic biosensor based on detection of changes in optical density on the surface of a narrow bandwidth guided mode resonant filter is demonstrated. Using sub-micron microreplication of a master sensor surface structure on continuous sheets of plastic film, the sensor can be produced inexpensively over large surface areas. In this work, the sensor structure is incorporated into standard 96-well microtiter plates and used to perform a protein–protein affinity assay. A surface receptor immobilization protocol demonstrating low nonspecific binding is used to detect an antibody with 8.3 nM sensitivity. By measuring the kinetic interaction of a protein–protein binding pair simultaneously at several concentrations, the affinity binding constant can be quickly determined.

Introduction

For the majority of assays currently performed for genomics, proteomics, pharmaceutical compound screening, and clinical diagnostic applications, fluorescent or colorimetric chemical labels are commonly attached to the molecules under study so they may be readily visualized [1], [2], [3]. Because attachment of a label substantially increases assay complexity and possibly alters the functionality of molecules through conformational modification or epitope blocking, various label-free biosensor technologies have emerged. Label-free detection phenomenologies include measuring changes in mass [4], microwave transmission line characteristics [5], microcantilever deflection [6], or optical density [7], [8] upon a surface that is activated with a receptor molecule with high affinity for a detected molecule. The widespread commercial acceptance of label-free biosensor technologies has been limited by their ability to provide high detection sensitivity and high detection parallelism in a format that is inexpensive to manufacture and package. For example, biosensors fabricated upon semiconductor or glass wafers in batch photolithography/etch/deposition processes are costly to produce and package if the sensor area is to be large enough to contain large numbers of parallel assays. Similarly, the requirement of making electrical connections to individual biosensors in an array poses difficult challenges in terms of package cost and compatibility with exposure of the sensor to fluids.

In previous work, we have described a novel technology based upon a narrow bandwidth guided mode resonant filter structure that has been optimized to perform as a biosensor [9]. The sensor utilizes a sub-wavelength grating waveguide structure to provide a surface that, when illuminated with white light at normal incidence, reflects only a very narrow (resonant) band of wavelengths. The resonantly reflected wavelength is modified by the attachment of biomolecules to the waveguide, so that small changes in surface optical density can be quantified without attachment of a label to the detected biomolecule. Unlike optical detection approaches that rely upon interaction of detected molecules with an evanescent wave, the detection phenomenon in this work actually occurs within the waveguide, and thus, provides for a strong interaction between surface binding events and the transduced signal. Further advantages of the sensor approach are that the resonant reflected signal is measurable with the sensor either dry or immersed in liquid, and the simplicity of the non-contact excitation/detection instrumentation. The sensor structure, when fabricated on a glass substrate, demonstrated surface binding sensitivity of 4.2×10⁻¹³ g/mm². Assays were performed that demonstrated the ability to perform a protein–protein binding assay with a sensitivity of 0.0167 nM for streptavidin, and the ability to measure attachment/detachment of molecules with molecular weight of 130 Da was demonstrated. Because sub-micron sensor feature definition and processing was performed on glass wafers in small batches, the sensor structure was limited in its use for low-cost disposable applications, such as microtiter plates or microarray slides.

In this work, we report on the design, fabrication, and evaluation of an equivalent sensor structure that is fabricated into sheets of plastic film. Rather than perform sub-micron definition of grating features using photolithography on the sensor itself, a master wafer is created in silicon that is used as a template for producing the sensor structure on plastic by a high-definition microreplication process. The use of a continuous plastic roll for the sensor substrate enables other processes, such as dielectric thin film coating and surface activation, to be performed in a substantially more efficient manner. Most importantly, the ability to produce a high-sensitivity biosensor in plastic over large surface areas enables incorporation of the sensor into large area disposable assay formats such as microtiter plates and microarray slides. In this work, we demonstrate incorporation of the plastic sensor into the bottoms of bottomless 96-well microtiter plates, and the ability to use the sensor plate to perform multiple protein–protein binding assays in parallel. The detection sensitivity of the plastic-substrate sensor is found to be equivalent to previously reported glass-substrate sensors.