Suppression of Bulk Fluorescence Noise by Combining Waveguide- Based Near-Field Excitation and Collection Md. Mahmud-Ul-Hasan,* ,†,‡ Pieter Neutens, ‡ Rita Vos, ‡ Liesbet Lagae, †,‡ and Pol Van Dorpe †,‡ † Department of Physics and Astronomy, KU Leuven, Celestijnenlaan 200D, 3001 Leuven, Belgium ‡ imec, Kapeldreef 75, 3001 Leuven, Belgium * S Supporting Information ABSTRACT: A high surface to bulk fluorescence ratio is very useful in bioimaging, sensing, sequencing, and physical chemistry character- ization. We used the evanescent field of a photonic waveguide for highly localized excitation and collection of molecular fluorescence. As both near-field excitation and collection are strongly distance dependent, we were able to increase the surface to bulk fluorescence ratio significantly. We have also experimentally investigated the combined excitation and collection efficiency as a function of the position of the molecule in the near field. Finally, we formulated and experimentally verified a general condition for the waveguide−molecule interaction length for maximum optical efficiency of the device. KEYWORDS: near-field excitation, near-field collection, waveguide sensor, bulk fluorescence, evanescent sensing F luorescence is widely used as the transduction medium for biosensing and physical chemistry characterization appli- cations. 1 A better surface to bulk fluorescence ratio is very useful for all these applications. In fluorescence-based surface biosensors, the target analyte molecules are captured on the surface. The emission from the fluorescently labeled captured molecules is detected and quantified to obtain the concen- tration. An improvement in surface to bulk ratio can help to speed up the measurement time in most current techniques, such as ELISA, FISH, next-generation DNA sequencing, and others by avoiding the washing steps currently needed. These washing steps are used to reduce the bulk fluorescence noise by removing the unbound fluorescent molecules floating in the bulk solution. A better surface to bulk ratio also means a shorter effective length along the vertical direction of the observation volume. Limiting the observation volume is useful for single- molecule fluorescence correlation spectroscopy to isolate a few molecules. 2,3 It is also instrumental for cell membrane studies. The evanescent field generated by the total internal reflection at the solid/liquid interface has been a good tool for surface−bulk separation. 4,5 The on-chip version for compact devices to generate an evanescent field has been the usage of the waveguide mode. 6 However, this is still far from an ideal situation, as there is a big mismatch between the typically used evanescent field that extends from 80 to 200 nm above the surface into the bulk 7 compared to the typical biosensing layer that ends within 10 nm. 8 We propose to use a photonic waveguide not only to excite the molecule in the near field but also to collect the subsequent emission in the near-field using the same waveguide. This way, both the excitation and collection efficiency have an exponential dependency on the molecule−waveguide distance. Hence, a big fraction of the bulk fluorescence generated during the evanescent excitation can be suppressed by distance-dependent near- field coupling. Although the scheme of excitation and collection by the same waveguide has been reported in a couple of publications on fluorescence 9 and Raman sensing, 10 the focus has been mainly on using the photonic waveguides as a mean of miniaturization for on-chip sensing applications. Here, we explore the possibility of using waveguides beyond the miniaturization aspect and rather use them as solution to the fundamental problem of background fluorescence noise in sensitive biosensing or single-molecule analysis. The experimental setup is illustrated in Figure 1. An input grating coupler has been used to couple 637 nm excitation light to a single-mode silicon nitride (SiN) strip waveguide. The input light excites the molecules residing within the evanescent field of the waveguide mode in the active sensor area. The subsequent emission from the molecules couples back to the waveguide. The fluorescence signal copropagating with the excitation signal is then coupled out of the chip using an output grating coupler. The light is collected and collimated by an objective. The collimated light passes through an appropriate filter set to the detector. We have investigated the excitation and collection efficiency as a function of position of the molecules in the near field above the waveguide. We deposited different thicknesses of SiO 2 spacer layers (10−400 nm) using a PECVD process at 350 °C on top of our pilot-line processed chip described in the Methods section. This spacer was used as a controllable separation between waveguide and solution. During this step, a Received: December 20, 2016 Published: February 9, 2017 Letter pubs.acs.org/journal/apchd5 © XXXX American Chemical Society A DOI: 10.1021/acsphotonics.6b01016 ACS Photonics XXXX, XXX, XXX−XXX