Integrated circuit microchip system with multiplex capillary electrophoresis module for DNA analysis

Analytica Chimica Acta 466 (2002) 187–192

Integrated circuit microchip system with multiplex capillary electrophoresis module for DNA analysis Joon Myong Song, Joel Mobley, Tuan Vo-Dinh∗ Advanced Biomedical Science and Technology Group, Life Sciences Division, Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, TN 37831-6101, USA Received 20 February 2002; received in revised form 12 June 2002; accepted 12 June 2002

Abstract In this paper, we describe the use of an integrated circuit (IC) microchip system as a detector in multiplex capillary electrophoresis (CE). This combination of multiplex capillary gel electrophoresis and the IC microchip technology represents a novel approach to DNA analysis on the microchip platform. Separation of DNA ladders using a multiplex CE microsystem of four capillaries was monitored simultaneously using the IC microchip system. The IC microchip–CE system has advantages such as low cost, rapid analysis, compactness, and multiplex capability, and has great potential as an alternative system to conventional capillary array gel electrophoresis systems based on charge-coupled device (CCD) detection. © 2002 Elsevier Science B.V. All rights reserved. Keywords: IC microchip; Capillary array electrophoresis; DNA separation; DNA ladder

1. Introduction DNA biosensors have been actively used as tools for recognizing and detecting specific target genes of biological and biomedical interest [1]. The use of gene probes as bioreceptors gives biosensors the ability to recognize and identify target genetic sequences in complex biological samples through specific interactions between the target genes and the bioreceptors, permitting, for example, highly specific disease diagnosis [2–4]. Beyond hybridization, the biosensors must also be able to detect the hybridized probe. This sensitive detection of target genes is paramount to successful biosensor design. Most sample platforms in DNA biosensors are based on fiber-optic probes or ∗ Corresponding author. Tel.: +1-865-574-6249; fax: +1-865-576-7651. E-mail address: [email protected] (T. Vo-Dinh).

silica plates and rely on fluorescence for detection. Due to its high sensitivity, the laser-induced fluorescence technique has been successfully applied in DNA biosensors for ultra-sensitive target gene detection [5], using conventional photomultiplier tubes and charge-coupled devices (CCDs) to detect emission of fluorescently-labeled target genes. Due to its multichannel capability, CCDs are very useful for high-throughput detection. However, CCD detection systems are relatively large, costly and overly complex and hence are mainly suitable for laboratory analysis. In order to overcome the size and cost limitations of CCDs, our laboratory has developed a novel biosensor based on integrated circuit (IC) microchips with complementary metal oxide semiconductor (CMOS) photocell arrays for optical detection [6,7]. This device is a miniaturized and integrated microchip system that includes photosensors, amplifiers, and logic circuits in a single IC package. This IC microchip

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is inexpensive and can provide a simultaneous, fast, and multifunctional analysis using microdot arrays with a variety of bioreceptors. Successful applications of this IC microchip to analysis using DNA probe microarrays have been reported [6,7]. A novel biomedical application of the IC microchip system involves its combination with the conventional DNA separation technique. Although the DNA probe microarray technique is highly selective, sequence information for target genes is required and each probe gene must be synthesized for each target gene. In contrast to a DNA probe microarray, the DNA separation technique can identify target genes based on DNA fragment sizes [8–11], making it unnecessary to prepare a set of gene probes, which, in turn, can reduce cost for analysis. In order to exploit the advantages of the IC microchip, the DNA separation system should be compact, high speed, and provide high-throughput. Over the past decade, capillary electrophoresis (CE) has been utilized as an alternative to slab gel electrophoresis in modern biology for separation of DNA fragments (e.g. polymerase chain reaction (PCR) products) with higher speed and separation resolution compared to slab gel electrophoresis. A variety of applications of CE to DNA sequencing [10,11], restriction digests [12], genotyping [13], and mutation analysis [14] using high speed and resolution have been reported. Based on the above advantages of CE, the multiplex capillary gel electrophoresis technique [15–19] fulfills those requirements for the IC microchip system, as it is well known for rapid separation and compactness. Also, the multiplex capability provides high-throughput analysis. In this report, we demonstrate multiplex capillary gel electrophoresis with an IC microchip detection system using laser-induced fluorescence. This work shows that the IC microchip system can be used as a highlycompact and inexpensive alternative to CCD detection for multiplex capillary gel electrophoresis. 2. Experimental 2.1. Fluorescence detection and IC microchip system Fig. 1 shows a schematic diagram of multiplex capillary gel electrophoresis/IC microchip system. For excitation, an Omnichrome-532 Argon-ion laser was used. The 514.5-nm laser line was selected using an

Fig. 1. Schematic diagram of the experimental apparatus.

equilateral prism. Extraneous light from the laser was removed by a pinhole. The laser beam was focused onto an outermost capillary of a four-capillary array. The laser power incident on the capillary array was 10 mW. Fluorescence from each capillary was collected using 5X microscope objective (Nikon, 0.1 NA) that was perpendicular to multiplex capillary and focused onto the detection elements on the IC microchip. Only four detection elements from a single column were used to detect the fluorescence from the four capillary arrays. A long-pass filter (cut-off position: 590 nm, Edmund Industrial Optics) was attached on the IC microchip to eliminate the laser scattering. The 5X microscope objective and long-pass filter are both compact, and thus are very suitable for the miniaturized IC microchip system. The IC microchip used in this work consists of a 4 × 4 array of photodiodes integrated into a single IC package along with signal processing and photodiode element addressing circuitry. The IC microchip was custom designed in our laboratory and fabricated using conventional CMOS techniques. The CMOS-based system can be operated using low supply voltages, and the CMOS production processes permit these chips to be manufactured inexpensively. The CMOS microchip detection elements were individually addressed and read out using digital I/O lines and an analog-to-digital conversion channel provided by a National Instruments DAQ516 PCMCIA card, installed in a laptop computer. The data acquisition

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process was controlled via a custom written software interface constructed with LabView. 2.2. Reagents and sample preparation 1XTris-Borate-EDTA (TBE) buffer solution was prepared by dissolving premixed powder in purified water according to procedure specified by the manufacturer (Amresco, Solon, OH). The composition of 1XTBE buffer was 89 mM Tris(hydroxymethyl)aminomethane (THAM), 89 mM Boric acid, and 2 mM ethylenediaminetetraacetic acid (EDTA). Rhodamine 610 solution was purchased from Exciton (Dayton, OH). The 100 bp DNA ladder (500 ␮g/ml) stock solution was obtained from New England Biolab. Inc. DNA sample solutions were prepared from dilution of the stock 100 bp DNA ladder solution with 1XTBE buffer. Capillaries (75 ␮m i.d., 365 ␮m o.d. and 50 ␮m i.d., 365 ␮m o.d.) were obtained from Polymicro Technologies (Phoenix, AZ). Capillary with 50 ␮m i.d. and 365 ␮m o.d. was used for the single-capillary system and capillary with 75 ␮m i.d. and 365 ␮m i.d. was used for the multiplex capillary system. Poly(vinyl pyrrolidone) (PVP) was purchased from Aldrich (Milwaukee, MI). PVP (Mr , 130,000) was used as a separation medium in this work. Its selection was based on successful DNA separations observed in previous studies [20]. The running buffer solution was 1XTBE with 0.5 ␮g/ml ethidium bromide. The separation medium was prepared by disso lving 1% PVP in 1XTBE buffer with 0.5 ␮g/ml ethidium bromide in a vial for 30 min with soft shaking. 2.3. Multiplex capillary gel electrophoresis The multiplex CE microsystem consisted of four capillaries, which were packed side by side at window region. Each window was made by burning off the polyimide coating of capillary. The windows were washed with methanol-soaked lens cleaning paper repeatedly before the capillaries were packed. A capillary holder mounted on a translational stage held the closely packed capillaries and the multiplex capillary was adjusted to be parallel to the optical bench. The separation medium was loaded into the given capillaries using a 100-␮l syringe. The ends of capillaries were immersed into running buffer solutions. Before DNA sample injection, an electric field of 120 V/cm

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was applied to the capillaries for 20 min to stabilize the separation medium. DNA sample solution was injected into the capillaries electrokinetically for 5 s at 10 kV. A high-voltage power supply (Hipotronics, model: R30B) was used to generate the electric field. After the run, the separation medium in each capillary was flushed out with water using a 100-␮l syringe. This process was repeated for each run.

3. Results and discussion The IC microchip consisted of 16 detection elements arranged in a 4 × 4 array. Each detection element was a CMOS-based photodiode with a square 900 ␮m × 900 ␮m aperture. The distance between the elements was 100 ␮m. The outer diameter of the capillaries was 365 ␮m. The detection elements on the IC microchip detected magnified images from the multiplexed capillaries in order to monitor the time-dependent DNA fragment signals. Since we are using four capillaries, the optics was adjusted to project each capillary’s fluorescence onto a single element of the IC microchip array. The optical coupling of the capillaries and the IC microchip involved eliminating cross-talk among the four channels while producing a uniform response across all four capillaries through laser beam focusing. The optical geometry for laser beam focusing for the IC microchip–CE system is shown in Fig. 1. Initially, in order to optimize laser beam focusing onto the multiplex capillary, the four capillaries, packed in a custom capillary holder, were filled with water before laser beam focusing. The laser beam was focused onto the first capillary by a plano convex lens with a 40-mm focal length. To achieve simultaneous laser beam focusing for the multiplex system, the laser beam was adjusted so that it passed uniformly through all four capillaries. This laser beam focusing approach is very suitable for our miniaturized IC microchip system due to compactness of the optical geometry. On the other hand, the confocal scanning optical geometry requires a larger optical space compared to our miniaturized IC microchip system. A particular advantage of our system is that the array detector precludes the need for a scanning optical system. We merely need to magnify the image of the capillary array to match the dimensions of the photodiode array. A solution to

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Fig. 2. Fluorescence of 10−6 M Rhodamine 610 detected on the detection elements of IC microchip optically coupled to the multiplex capillary.

achieve magnified fluorescence images was to simply use a microscope objective. This microscope objective is compact and less expensive in comparison to other optical elements such as camera lens. Therefore, a 5X microscope objective was used in the present system to project four fluorescence images from the four channels of the capillary array onto the four detection elements on IC microchip. Fig. 2 shows the fluorescence intensities detected on the IC microchip when the four capillaries were filled with Rhodamine 610 solution at a concentration of 1 × 10−6 M with the IC microchip set to the lowest gain level. The bottom plane in Fig. 2 corresponds to the address of detection elements on the IC microchip. Each detection element is independently addressed by its column and row number. The vertical axis is the IC microchip signal in volts and is proportional to the emission intensity. The IC microchip set-up was designed to move in three dimensions with translational stages for alignment with the optimized focal plane of four fluorescence images. Only detection elements of column number 2 exhibited strong detection of the fluorescence as compared with the

other three columns. This result reflects the optical isolation of the four fluorescence images obtained from the capillaries. On the other hand, as the number of capillaries that the laser beam passes through increases, fluorescence intensities of Rhodamine 610 decrease. This condition is evident through the decreasing signal trend observed from row 4 to row 1. The highest overall fluorescence signal was obtained by optimizing the alignment of the laser beam with the multiplex capillary and by fine adjustment of the IC microchip position via translational stages. Following this optimization procedure, DNA separation was monitored directly on the IC microchip. We selected 100-base pair (bp) DNA ladders as a test sample for the separation of DNA fragments. In biomedical research, DNA ladders are usually used as DNA sizing markers for PCR techniques applied to areas such as genotyping and mutation analysis. The efficacy of the PCR reaction is determined by the degree of matching of the sizes between the DNA ladders and the PCR products. Accordingly, monitoring separation of DNA ladders on the IC microchip is a useful assay that will lead to successful applications of the

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Fig. 3. Electropherogram of 100 bp DNA ladders separated in single capillary with 50 ␮m i.d. and 365 ␮m o.d. The IC microchip was operated with 100 gain and 5 Hz data acquisition rate. DNA ladders were separated in 1% PVP sieving matrix. All peaks are assigned by the migration time. (1) 100 bp; (2) 200 bp; (3) 300 bp; (4) 400 bp; (5) 500 bp; (6) 600 bp; (7) 700 bp; (8) 800 bp; (9) 900 bp; (10) 1000 bp; (11) 1200 bp and (12) 1517 bp.

IC microchip–CE system to genotyping and mutation analysis. Fig. 3 shows the separation of 100 bp DNA ladders in a single capillary (50 ␮m i.d., 365 ␮m o.d.) obtained with the IC microchip. Electrokinetic injection of DNA ladders with 25 ␮g/ml was performed for 5 s at 10 kV. The separation electric field and effective separation length was 120 V/cm and 55 cm, respectively. The 1% PVP was used as a separation medium. Efficient separation of DNA fragments in CE is primarily dependent on the gel matrix that has the

appropriate mesh size providing DNA fragments with a suitable sieving effect. Using 1% PVP and an electric field of 120 V/cm, separations were performed on 12 DNA ladder samples. Up to 1000 bp, all DNA ladders were baseline separated, except for the 900 and 100 bp fragments. Also, 1200 and 1517 bp could be identified clearly. This separation performance demonstrates the great potential of the integrated CE–IC microchip system for biomedical applications such as genotyping and mutation analysis.

Fig. 4. Electropherograms of the four capillaries obtained with IC microchip–CE system. The labeled peaks correspond to the number of base pairs in the fragments as follows: (1) 100 bp; (2) 200 bp; (3) 300 bp; (4) 400 bp; (5) 500 bp; (6) 600 bp; (7) 700 bp; (8) and (9) 800, 900, 1000, 1200, 1517 bp.

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Fig. 4 shows four electropherograms of 100 bp DNA ladders that were simultaneously obtained using the four-capillary array system. The separation conditions of DNA ladders were similar to those applied in the single capillary trial described above. The concentration of the DNA sample was 75 ␮g/ml and was injected into the capillaries electrokinetically for 5 s at 10 kV. Each electropherogram was obtained at a data acquisition rate of 5 Hz. From 100 to 700 bp, DNA fragments could be identified, but DNA fragments larger than 700 bp came out without clear separation. Compared to the separations using a single capillary, the resolution of DNA ladder separations using the multiplex capillary system was reduced. This condition was mainly due to the larger diffusion of DNA fragments in the capillary with larger inner diameter. Note that separation of DNA fragments larger than 800 bp can be further improved through optimization of the electric field and gel types and conditions [9], however, such optimizations are beyond the objective of this work. Migration times of DNA fragments showed small changes from capillary to capillary due to inhomogeneities in the gel matrices, inhomogeneities in wall conditions, and inhomogeneities in applied electric fields. 4. Conclusion In this study, we demonstrated the feasibility of an IC microchip–CE system for multiplex analysis of four capillaries for DNA fragment analysis. The developments of advanced systems with a larger number of capillaries are being investigated. This miniaturized IC microchip–CE system using CMOS-based sensing arrays has several advantages over conventional multiplex capillary gel electrophoresis using CCD detection, including compactness, low detector voltage, and low cost. The data presented here show that the miniaturized IC microchip–CE system is a robust platform for DNA separation studies, and that it should be applicable to biomedical research such as genotyping, drug screening, and mutation analysis. Acknowledgements This work was sponsored by the Laboratory Directed Research and Development Program

(Advanced Nanosystems Project), Oak Ridge National Laboratory, by the US Department of Energy (DOE) Office of Chemical and Biological National Security and the DOE Office of Biological and Environmental Research, under contract DEAC05-00OR22725 with UT-Batelle, LLC. J.M. Song and J. Mobley are supported by an appointment to the Oak Ridge National Laboratory Postdoctoral Research Program administered jointly by the Oak Ridge Institute for Science and Education and the Oak Ridge National Laboratory.

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