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DNA Microarray Scanner with a DVD Pick-Up Head Available online at www.sciencedirect.com Current Applied Physics 8 (2008) 687–691 www.elsevier.com/locate/cap www.kps.or.kr DNA microarray scanner with a DVD pick-up head Kyung-Ho Kim a, Seung-Yop Lee a,*, Sookyung Kim b, Seong-Gab Jeong c a Department of Mechanical Engineering, Sogang University, Seoul 121-742, Republic of Korea b Nanostorage Co. Ltd., Seoul 120-090, Republic of Korea c Department of Mechantronics, Cheongju College of Korea Polytechnic IV, Cheongju, Chungbuk 36-290, Republic of Korea Received 29 July 2006; received in revised form 9 April 2007; accepted 27 April 2007 Available online 10 October 2007 Abstract A low-cost highly sensitive DNA microarray scanner for fluorescent detection is developed based on the pick-up head of a commer- cially available optical storage device, DVD. A laser beam of 650 nm, generated by a DVD laser diode, is used for dynamic auto-focusing as well as the excitation of Cy5 fluorescent dye. The fluorescence intensity emitted from Cy5 dye is measured by a photomultiplier tube (PMT). In contrast to other microarray scanners, the DVD-based scanner offers the auto-focusing function using the focus error signal (FES) and a voice coil motor (VCM), and this enables fast response, high accuracy and compact size. The fluorescence-detecting per- formance of the scanner is inspected by using a commercial BAC (bacterial artificial chromosome) oligonucleotide chip and a scanner evaluation microarray (DS01). Experiments have shown that the DVD-based scanner meets the limit of detection, ensuring the feasibility of a low-cost, highly sensitive DNA microarray scanner. Ó 2007 Elsevier B.V. All rights reserved. PACS: 42.62.Be; 42.79.Vb; 87.62.+n; 87.80.Tq Keywords: DNA microarray; DNA chip; Biochip; DVD; Fluorescent scanner; Auto-focusing actuator 1. Introduction Various researches have used optical storage technology as the detection mechanism for DNA microarrays. It was Today, most DNA or protein microarrays use fluorescent shown that CD technology could build a detection system dyes as a DNA marker. In general, the fluorescence-detect- with equivalent or even better performance than conven- ing technology has shortcomings, such as weak fluorescent tional scanners [3], and a lab on a chip (LOC) system has light and a highly controlled positioning mechanism. It is been developed for application in DNA analysis using a known that mechanical precision to 1 lm or less and a cool- CD disk [4,5]. ing device are required to meet the measurement specifica- tions of commercial confocal scanners. The high cost of 2. Auto-focusing technology the detection mechanism prevents a wide spread in the DNA-chip market, especially when high-density diagnostic When scanning a large surface area of a DNA micro- chips are used. Therefore, detection technology satisfying array, it is important to keep the distance between the both high sensitivity and low-cost becomes one of the essen- objective lens and the microarray surface constant with tial factors to expand the DNA-chip market and the related highest accuracy. The main advantage of the DVD pick- technology and industry [1,2]. up-based fluorescent detection is to easily implement a dynamic auto-focusing mechanism by attaching a reflective layer on the commercial microarray. Fig. 1 shows the func- * Corresponding author. Tel.: +82 2 705 8638; fax: +82 2 712 0977. tional difference between a conventional single point E-mail address: [email protected] (S.-Y. Lee). method and an auto-focusing control. Fig. 1a shows that 1567-1739/$ - see front matter Ó 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.cap.2007.04.047 688 K.-H. Kim et al. / Current Applied Physics 8 (2008) 687–691 Fig. 1. Comparison of (a) single point method and (b) auto-focusing method for misaligned or warped microarrays. Fig. 2. (a) Schematic diagram of optical path and (b) DNA microarray with a reflective layer. K.-H. Kim et al. / Current Applied Physics 8 (2008) 687–691 689 a misaligned or warped microarray can lead to unfocused The tracking motion is actuated and controlled by a precise images when the single point method is used. However, positioning stage. Fig. 1b shows the main advantage of the auto-focusing In order to implement the dynamic auto-focusing func- method, which compensates the mechanical tolerance of tion, a new microarray slide structure is designed and man- the DNA-chip surface. In general, the relative position of ufactured by attaching a reflective layer on a commercial the objective lens and the microarray surface is determined DNA microarray slide. Fig. 2b shows the slide structure by 5° of freedom (three translations and two rotations). consisting of two different layers. Since the DNA probe For the precise detection of the fluorescent marker, the sur- surface is directly attached on the reflective layer, the laser face translation motions (X and Y) must be less than 1 lm. beam is focused on the microarray surface and the reflec- A DVD optical pick-up, which is widely used in commer- tive layer simultaneously. cial optical storage devices, enables a low-cost sub-micron focusing control [6,7]. 4. Experimental results 3. Fluorescent detection mechanism Fig. 3 shows the experimental system to test the fluores- cence-detecting performance of the proposed DNA micro- Fig. 2a shows a schematic diagram of the optical paths array scanner. The focusing motion of the objective lens is of the proposed system. There are two different optical automatically controlled and the focal point falls upon the paths: one is for auto-focusing and the other is for fluores- spot center. The tracking motion is actuated by a precise cent detection. A laser diode generates a laser beam of motorized stage to scan the microarray surface. In order wavelength 650 nm. Most of the laser beam is reflected to increase the fluorescence signal, the signal-to-noise ratio by a dichroic filter (Chroma Optics Z660RDC), and the (SNR) is to be improved. To determine SNR, the following part of the laser beam passing through the dichroic filter formula is commonly used: is used to regulate the laser power by a front monitor sen- sor. The laser beam transmitted through the dichroic filter is focused on the reflective layer, and the reflected beam is finally focused onto a four-quadrant photodiode (PD) after passing through two dichroic filters. The focus error signal is calculated by using the intensities of the four divided regions of the PD. The PD generates a focus error signal whose magnitude is dependent upon the distribution of the beam spot across its four divided regions. The resulting servo signal is used to drive the VCM actuator in such a way that the objective lens is shifted to a point where its focal point falls upon the spot center. The laser beam focused on the DNA-chip surface excites Cy5 dye with DNA probes, emitting a fluorescent signal of 670 nm. The collimated emission beam is then refocused by a detec- tion lens and passes through a pinhole into the photomul- tiplier tube (PMT). The analogue electric signal from the Fig. 4. Measurements of signal-to-noise ratio (SNR) of two cases: with/ PMT represents the intensity of the fluorescent signal. without auto-focusing. Fig. 3. Experimental set-up of fluorescence-detecting mechanism with a DVD pick-up head. 690 K.-H. Kim et al. / Current Applied Physics 8 (2008) 687–691 Signal intensity À Background intensity are painted on a slide. Then, SNR was measured along the SNR ¼ : ð1Þ Standard deviation of background spot centers with constant fluorescent intensities after adjusting the initial position and light intensity on the spot Eq. (1) implies that one may increase the signal or/and re- center. For the auto-focusing case, the magnitude and uni- duce the background in order to improve SNR. Typically, formity of SNR are improved, as shown in Fig. 4. The ele- the limit of detection (LOD) is defined as the minimum vation of SNR is due to the uniform background by the detectable signal for which the SNR is 3. The sensitivity dynamic auto-focusing, the increase of fluorescent signal of a DNA-chip scanner is inversely related to the limit of by the reflective layer, and the decrease of noise. The weak detection – the detection sensitivity increases as the limit fluorescent level without auto-focusing is below the limit of of detection decreases. detection, and it does not guarantee the minimum detect- Fig. 4 shows a comparison of SNR for the two cases able signals. Thus, the dynamic auto-focusing mechanism with and without auto-focusing function. In the experi- implemented by the DVD pick-up system is required for ment, regular line patterns with constant dye concentration the scanner to meet the minimum fluorescence level. The Fig. 5. Experimental results using auto-focusing control (a) with auto-focusing and (b) without auto-focusing. Fig. 6. Detection images of evaluation slide (a) with auto-focusing and (b) without auto-focusing. K.-H. Kim et al. / Current Applied Physics 8 (2008) 687–691 691 use of the dynamic auto-focusing and reflective layer can pick-up. A laser beam of 650 nm, generated by a DVD remarkably improve the detection performance. In con- laser diode, is used for the dynamic auto-focusing as well trast, the non-uniform profile of SNR without auto-focus- as the excitation of Cy5 dye. The use of the dynamic ing is mainly caused by the background noise, surface auto-focusing and the reflective layer attached on DNA- variability, and misaligned slide surface. chip slides improves the sensitivity of fluorescent detection. Fig.
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