Combined Enhanced Fluorescence and Label-Free Biomolecular Sensing with a Two-Dimensional Photonic Crystal

A class of surface photonic crystals (PCs) termed guided mode resonant filters are capable of producing narrowband reflections that can be designed for optical wavelengths of choice by altering structure feature sizes. Since the observed narrowband reflection location is strongly modulated by the effective refractive index of the PC and surrounding environment, label-free biomolecular sensing can be accomplished by tracking reflection peak movements as proteins are deposited on the sensor surface. Additionally, these resonant phenomena are associated with high intensity near-fields that can strongly excite fluorophores in proximity to the photonic crystal surface. One-dimensional and two-dimensional PCs have previously been fabricated to perform label-free sensing of biomolecules as well as to enhance the signal measured from organic fluorophores]. By performing both enhanced fluorescence and label- free molecular detection on a single PC, important life sciences experiments such as cell-based assays and DNA microarrays can be performed with greater accuracy and higher sensitivity. Our work describes the first photonic crystal designed to perform enhanced fluorescence and label-free sensing with a single surface area. A two-dimensional photonic crystal with different periods in each lateral direction (Figure 1) was designed using Rigorous Coupled Wave Analysis to exhibit resonant peaks at two distinct wavelengths. The PC is designed such that one peak is excited by polarized light in the visible (633 nm), near a laser line commonly used to excite the organic fluorophore Cyanine-5 (Figure 2a). Simulations show a high intensity near- fields are present at resonance, increasing the emitted intensity from fluorophores at the PC surface. The other peak is designed such that it is excited with an orthogonal polarization of light in the near-infrared (850 nm), so that reflection spectra can be obtained with a commercially available imaging spectrometer-based instrument (Figure 2b). Label-free images of molecular binding can be obtained by measuring the resonant wavelength of the PC surface as a function of position, where absorption of biomolecules leads to shifts in the resonant wavlength. An 8-inch diameter silicon master wafer with a negative image of the desired PC surface structure is fabricated using deep-UV lithography. From this master, the device is replica molded into UV-curable polymer, and TiO₂ is sputtered onto the surface (Figure 3). This process creates sensors on plastic sheets over large areas that can be adapted to single-use disposable labware such as microtiter plates and microscope slides. The reflection peaks observed when illuminating the structure with two different polarizations of white light correlate with those predicted by simulation. Spots of Cyanine-5-conjugated streptavidin (500 mum in diameter) are then applied to the sensor and can be compared with a reference surface. Upon imaging the spotted PC sensor with a commercially available microarray scanner, spots on the device have exhibited as much as a 500x increase in fluorescence intensity as compared with spots that were not present on the PC (Figure 4). The same streptavidin spots can be imaged using the imaging spectrometer-based instrument, as shown in Figure 5.