Exploring Optical Properties of Liquid Crystals for Developing Label-Free and High-Throughput Microfluidic Immunoassays By Chang-Ying Xue, Saif A. Khan, and Kun-Lin Yang* Immunoassays developed in microfluidic systems have attracted a lot of attention in recent years, because they require small sample volumes and have faster reaction times. [1–8] However, the major challenge in miniaturizing current microfluidic immu- noassays into useful lab-on-chip devices is the detection mechanism. Since most microfluidic systems still rely heavily on enzyme-catalyzed reactions (e.g., enzyme-linked immunosor- bent assays, ELISA) or fluorescence (e.g., immunofluorescence assays) for detection, [9–14] the use of bulky equipment such as spectrometers or fluorescent microscopes preclude the use of microfluidic immunoassays for point-of-care (POC) applications. Furthermore, labeling antibodies with enzymes or fluorescence probes requires additional preparation steps, which further limit the possibility of preparing a fully integrated system. To address this issue, we report a label-free detection mechanism based on the interactions between liquid crystals (LCs) and antibodies. We show that by covering microfluidic immunoassays with a thin layer of liquid crystal, antibody concentrations can be quantified in the length of the bright LC region in the microfluidic channels, and test results can be readily observed with the naked eye, without any instrumentation. To identify suitable experimental conditions for developing a LC-based microfluidic immunoassay, we first prepared a fluorescence immunoassay in a microfluidic device, as shown in Figure 1. This microfluidic device comprises three serpentine channels supported on an immunoglobulin (IgG)-coated glass slide and sealed with poly(dimethylsiloxane) (PDMS) with embedded microfluidic channels. Two fluorescence-labeled antibody solutions, fluorescein-isothiocyanate-conjugated anti- human IgG (FITC-anti-IgG) and FITC-conjugated anti-biotin (FITC-anti-biotin), were then pipetted into the inlet reservoirs of two separate channels, allowing both solutions to enter the microfluidic channels by capillary action. After rinsing and drying, fluorescence images as shown in Figure 2a were obtained. We can draw several conclusions from Figure 2a. First, green fluorescence only appeared in the FITC-anti-IgG channel, suggesting that the binding of FITC-anti-IgG to IgG is highly specific. In contrast, no fluorescence was observed in the FITC-anti-biotin channel, indicating that very little or no nonspecific adsorption occurred on the surface. Second, the green fluorescence decreases continuously in the FITC-anti-IgG channel, indicating that the experimental conditions employed here allow the concentration of FITC-anti-IgG to deplete in the channel, leading to a concentration gradient in this channel. We also conducted another similar experiment with a biotin-labeled bovine serum albumin (bi-BSA)-coated surface. As expected, after flowing FITC-anti-IgG and FITC-anti-biotin into two different channels, only the FITC-anti-biotin channel showed green fluorescence with a gradient (Fig. 2b). Further inspection of Figure 2a and b also reveals that the fluorescence in the FITC-anti-biotin channel decreases more rapidly than that in the FITC-anti-IgG channel (Fig. 2c). This phenomenon could be explained by a difference in their binding rates. We can define a characteristic binding time t for both antigen-antibody pairs as follows: [15] t ¼ 1 k on C þ k off (1) COMMUNICATION www.advmat.de Figure 1. a) Schematic illustration of a LC-based microfluidic immunoas- say. The surface of the glass slide is coated with a layer of antigen (IgG or bi-BSA). Buffer solutions containing different antibodies (without labeling) can be pipetted into the inlet reservoirs (indicated by arrows), allowing them to enter the microfluidic channels by capillary action. After incubation and rinsing, a thin layer of LCs is supported on the glass slide, to report the result of the immunoassay. b) Cross-sectional SEM image of the micro- fluidic immunoassay, showing detailed dimensions of the microfluidic channels (width  depth ¼ 200 mm  160 mm). [*] C. Xue, S. A. Khan, Prof. K. Yang Department of Chemical and Biomolecular Engineering National University of Singapore 4 Engineering Drive 4, Singapore 117576 (Singapore) E-mail: cheyk@nus.edu.sg DOI: 10.1002/adma.200801803 198 ß 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Adv. Mater. 2009, 21, 198–202