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Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy Sensors and Actuators B 169 (2012) 144–150 Contents lists available at SciVerse ScienceDirect Sensors and Actuators B: Chemical j ournal homepage: www.elsevier.com/locate/snb Double spiral detection channel for on-chip chemiluminescence detection a a b,∗ Khoi Seng Lok , Yien Chian Kwok , Nam-Trung Nguyen a National Institute of Education, Nanyang Technological University, Nanyang Walk, Singapore 637616, Singapore b School of Mechanical and Production Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798, Singapore a r t i c l e i n f o a b s t r a c t Article history: In this paper, we introduced a three-layered microchip that consists of a double spiral channel design Received 12 April 2011 for chemiluminescence (CL) detection and a passive micromixer to facilitate the mixing of reagents. Received in revised form 18 April 2012 The design with two overlapping spiral channels doubled the CL intensity emitted from the reaction as Accepted 19 April 2012 compared to the design with a single spiral channel. The addition of the passive micromixer improved Available online 27 April 2012 the signal by 1.5 times. Luminol-based chemiluminescence reaction was used for the characterization of the device. This microchip was successfully tested with the determination of l-cysteine and uric acid. Keywords: © 2012 Elsevier B.V. All rights reserved. Chemiluminescence Lab-on-chip Micro total analysis system Luminol Cobalt l-Cysteine Uric acid 1. Introduction intensity of the signal is not as bright as in fluorescence, in which there is an excitation light source. Therefore, a sensitive optical Chemiluminescence (CL) detection methods do not require an detection system such as a standard fluorometer or a photon- excitation light source. Hence, compared to fluorescence detec- multiplier tube (PMT) is needed to capture this immediate light tion where an excitation source is required, CL detection could emission. These drawbacks posed many challenges in implement- be realized in a more compact system for portable applications. ing CL detection system in ␮TAS/LoC devices. Such systems are often called micro total analysis system (␮TAS) Micromixers are used in ␮TAS/LoC devices to assist mixing or labs on a chip (LoC). The CL signal solely emits from the chemical of fluids in the microchannels. Simple serpentine mixing chan- reaction. There are limited molecules species that are chemilumi- nels were often used in the microchip designs [3–5]. Mixing in nescent. Consequently, background interferences can be reduced. these devices relied entirely on molecular diffusion of the reac- There are many CL systems such as luminol, acridinium com- tants. The flow rate needs to be kept at a low value to minimize pounds, coelenterazine, dioxetances and luciferase. These systems convective transport. Mei et al. showed that having staggered have various applications in immunoassays, receptor assays, DNA herringbone micromixer (SHM) in the microchip improved the probes and biosensors [1]. Mangru and Harrison first introduced sensitivity of luciferase assay by three times [6]. Hence, having a the use of CL detection in microchip-based capillary electrophore- proper micromixer design in the microchip may be important to sis, in which horseradish peroxidase catalyzed reaction of luminol facilitate maximum mixing efficiency, followed by maximum reac- with peroxide was used as a post-separation detection scheme [2]. tion efficiency prior to the detection of CL signal. The length of the Following this, CL detection was popular in various LoC applica- micromixer is also important to improve the quality of fluid mixing. tions. According to Lin’s work [7], a similar SHM requires 60 mm in length The main drawbacks of CL reactions are the rapid reaction time to achieve a mixing efficiency of 95%. Williams et al. also gave an and the fast decay of the emitted luminescent signal. Hence, the in-depth analysis of a SHM, in which mixing was found to be a func- reactants need to be mixed quickly, followed immediately by its tion of Peclet number in the mixer [8]. Lok et al. showed that the detection. The signal is emitted solely from the reaction. Hence, the length for a reaction channel is longer than the mixing channel for luminol CL assay [9]. This extended length needed is likely due to the delay in CL emission. ∗ In flow injection analysis (FIA), the flow cell is coupled close Corresponding author. Tel.: +65 6790 4457; fax: +65 6792 4062. to the photon detector. Nozaki et al. first introduced a setup of E-mail addresses: [email protected] (Y.C. Kwok), a coiled Teflon tube packed horseradish peroxidase-immobilized [email protected] (N.-T. Nguyen). 0925-4005/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2012.04.047 Author's personal copy K.S. Lok et al. / Sensors and Actuators B 169 (2012) 144–150 145 gels in front of a PMT for the fast determination of hydrogen per- oxide [10]. Kiba et al. expanded this idea to a silicon-glass flow cell containing spiral channel for the similar chemistry [11]. Zhang et al. also introduced another variant of the spiral flow cell design [12]. Terry et al. created an intricate sinusoidal channel for better cou- pling to photon detector [13]. These works aims to maximize CL detection in flow cell. There are rooms for improvement by having a better micromixer and maximizing the area exposed to the photon detector in ␮ TAS/LoC. The light intensity from the CL reactions is dependent on the amount of excited molecules. Therefore, this intensity can be increased by increasing the concentration of reactants in a given volume or increasing the reaction volume at a given concentration of reactants. A stronger light signal can be generated by using a larger reaction volume. Using these principles, the reaction volume exposed to the photon detector can be increased by overlapping two spiral channels. The two spiral channels can be fabricated on two layers of the same microchip, which they are connected by a hole. Further improvements can be achieved by having a mirror to reflect the scattered light back to the detector and a more sensitive detector to record the weak signal. Additional setup such as lenses, optical fiber and photonic crystal fiber [14] could also be used to enhance the transfer of the emitted light to the detector. In this paper, we used a luminol-based CL system for detection purpose. CL with luminol (5-aminophthalhydrazide) is a well- characterized reaction system that have been useful for various applications such as monitoring of metals and other pollutants in water [15,16], immunoassays, DNA analysis [17] and dating of human remains [18]. During this reaction, luminol is oxidized to 3- aminophthalate ions with the aid of a catalyst or co-oxidant under alkaline aqueous conditions, producing water, nitrogen gas and light (wavelength = 425 nm). Cobalt(II) ions are known to be more effective catalysts for this reaction compared to Cu(II), Ni(II), Fe(III), Mn(II) and Fe(II) ions [19]. The aim of the present study is first to verify that a double spi- Fig. 1. Schematic layout of the microchips design A, B and C. Design A consists of ral channel design would improve the strength of the CL intensity a double spiral channel system which overlaid each other to provide a full circular emitted from the reaction. Second, a passive micromixer is added detection volume of 25 mm in diameter. Design B consists of a passive micromixer to this double spiral channel design further improving the CL sig- and a single spiral detection channel. Design C consists of a passive micromixer and l nal. -Cysteine and uric acid determination are demonstrated in the a double spiral channel system. new microchip design. 2. Materials and methods 2.1. Microchip design Three microchip designs A, B and C were investigated in this work (Fig. 1). The devices consisted of three layers. These microchips had three inlets to provide versatile reagents input into the system. Design A consisted of a double spiral channel to maximize area for optical detection. Design A measured 40 mm in length, 30 mm in width and 3 mm in thickness. Design B consisted of a passive micromixer for fluids mixing and a single spiral design for optical detection. Design C consisted of a passive micromixer for fluid mix- ing and a double spiral design for optical detection. Both designs B and C measured 50 mm in length, 30 mm in width and 3 mm in thickness. The purpose of having design A and C was to verify the effect of having a micromixer on the efficacy of the CL reaction. The purpose of having design B and C was to evaluate the sensitivity of single spiral channel and double spiral for optical detection. The double spiral design was formed by a clock-wise spiral channel in the middle layer, which was overlapped by an anti- Fig. 2. Three-dimensional schematic layout of the microchip design A, containing clockwise spiral channel in the bottom layer.