Keywords

Metal-Enhanced Fluorescence, Glancing Angle Deposition, Microarray

1. Introduction

Microarray bio-chips, including DNA, protein, and antibody microarrays, are essential tools for high-throughput analysis of biochemical reactions [1–3]. Among the various detection methods, fluorescence-based detection techniques are widely used in microarray bio-chips because of their fast detection, good repeatability, and non-radiative nature [4]. Although a high reliability had been reported for conventional fluorescence microarray analyses at high molecular concentrations, a large variation in the fluorescence detection at low molecular concentrations due to low sensitivity was reported [5,6]. Metal-enhanced fluorescence (MEF) is a powerful technique to improve the sensitivity of fluorescence-based analyses by allowing the fluorophores to interact with the enhanced electromagnetic fields generated by the localized surface plasmon resonance (LSPR) effects of metallic nanostructures [7]. Extensive literature has reported the effects of metallic nanostructures, with various shapes and configurations, on the enhancement of fluorescence by the LSPR effect [8–12]. Pompa et al. [7] fabricated gold nanostructure by electron beam (E-beam) lithography technique for MEF substrate and achieved the enhancement factor of ~ 30x. However, the E-beam lithography technique is limited in application to microarray bio-chips requiring a large-area MEF substrate (typically slide glass size, 25 × 75 mm2) owing to its high processing cost and long processing time. Glancing angle deposition (GLAD), a well-known single-step large-area fabrication technique for nanostructures, has been applied to fabricate large-area MEF substrates. GLAD is a physical vapor deposition process in which deposition flux is incident onto the substrate at a large deposition angle with regard to the surface normal (approximately 75°–90°) [13–16]. It is a simple fabrication process, providing a columnar morphology, using the “shadowing effect” and precise in-plain rotation of the substrate. In the early stage of the GLAD process, the deposited particles are randomly scattered, forming island structures on the substrate. As the deposition proceeds, the initial nucleated islands act as shadowing centers, with taller islands receiving more impinging particles than the shorter ones (shadowing effect). This competition causes the taller islands to grow into columns, resulting in the formation of nanorod structures [17–19]. Abdulhalim et al. [20] fabricated tilted nanorods with different materials using GLAD for MEF and concluded that the Ag nanorods show the highest enhancement factor compared to copper, gold, and silicon. Ju et al. [21] and Dahuruv et al. [22] reported 23× and 32× enhancement factors, respectively, using tilted (slanted) GLAD nanorod structures. Badshah et al. [23] reported a 200× enhancement factor using vertical GLAD nanorod structures (with a height of 500 nm) for DNA microarray application, in which the large enhancement factor was due to the LSPR effect of GLAD nanorod and the increase of binding property of fluorophore because the fluorescence signals were compared after the washing process. Although the fluorescence signal of the fluorophore at the spotting area was significantly increased in the GLAD-nanostructured MEF substrate, the signal-to-background noise ratio (SBR) of the substrate was not increased significantly as the background noise at the non-spotting area (except the bio-receptor spotting area) was also enhanced due to the MEF effect. In the microarray bio-chip, the background noise emanating from the nonspecific absorption of materials at the non-spotting area [24,25]. As the background signal can be a threshold signal for detection or identification in microarray analysis [26,27], a method to minimize the background signal and increase the SBR would be required in the MEF substrate for it to be applicable as a microarray bio-chip. To achieve the high SBR on MEF substrate, a selective fabrication of metallic nanostructure only on the spotting area is required.
In this study, we proposed an Ag nanorods on micropost array (AgNMPA) MEF substrate to improve SBR, which was fabricated by GLAD process of Ag nanorods on the UV-imprinted micropost array. During the GLAD process, the Ag nanorods were formed only on top of the micropost array because of the shadowing effect of the pre-existing micropost structures [28]. Figure 1 shows the schematic representation of the proposed AgNMPA MEF substrate and its working principle to reduce the background noise. The background noise can be reduced in two ways: first, the fluorescence signal is enhanced only on top of the micropost structures (spotting area), because the GLAD Ag nanorods are selectively formed only on top of the micropost structures. Second, in the fluorescence signal measurement using the confocal microarray scanner system, only the top of the micropost structures (spotting area) can be imaged, whereas the non-spotting area cannot, as the latter is located at an out-of-focus region. In addition, the proposed AgNMPA MEF substrate can provide uniform size of spotted antibody due to the restriction of spot spreading on the top micropost, which cannot be achieved in the GLAD Ag nanorod MEF substrate due to the randomized capillary force of nanostructure.
To fabricate the proposed AgNMPA MEF substrates, we used a silicon master containing a micropost array with a pitch of 600 µm, diameter of 300 µm, and height of 50 µm. A polymer micropost array, with dimensions similar to those of the silicon master, was obtained by the UV-imprinting process on the entire area of the conventional glass slide substrate using polydimethylsiloxane (PDMS) mold replicated from the silicon master. As the configuration of the Ag nanorods on the top and bottom of micropost substrate can be affected by the deposition angle of the GLAD process, the vertical GLAD nanostructures with various deposition angles (81° to 89°) were fabricated on the UV-imprinted micropost array substrate. To examine the performance of the fabricated AgNMPA MEF substrates, we spotted the human myeloid progenitor inhibitory factor 1(MPIF-1) capture antibody on the top of the micropost using an aligned spotting system and detected the MPIF antigen with Cy-3 fluorescence dye. Finally, the performance of the AgNMPA MEF substrate was tested and compared to that of the GLAD MEF substrate and conventional glass substrate.