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A ZEPTO MOLE DNA MICRO SENSOR* Tza-Huei Wang, Sylvain Masset, and Chih-Ming Ho Mechanical & Aerospace Engineering, University of California Los Angeles, CA 90095-8357, USA With the introduction of molecular beacon, DNA detection with high specificity can be performed directly in a microchannel (inchannel detection) that reduces the necessary sample volume by several orders of magnitude compared with most of the other DNA sensors [3,4] and also simplifies the processes of integrating a biosensor into a micro total analysis system(µ-TAS). In this paper, microchannels with different crosssection geometries were fabricated and their sensitivity of DNA detection were compared to determine the optimum design. Metals with high reflectance like Al and Au were deposited and patterned to form mirror-like sidewalls on the detection region of the channels to enhance the surface reflectance and increase the fluorescence signal level. ABSTRACT Using molecular beacons (MB) as highly sensitive and selective nucleic acid probes, we eliminate two of the major but cumbersome steps, probe immobilization and washing, of genebased biosensors. By integrating sidewall mirrors with microchannels, we can increase the signal-to-noise ratio of the optical detection. The detection specificity is achieved using a MB based DNA hybridization technique. Molecular beacons become fluorescent only upon hybridization with target DNA/RNA molecules as the quencher is separated from the fluorophore. We can hence eliminate washing and immo bilization steps and establish the inchannel detection technique. This technique reduces the detection volume to 36 pL. Microchannels coated with metal films with high reflectance are used to increase the signal level in the Laser Induced Fluorescence(LIF) system. By using the side-mirror channel with inchannel sensing technique, the detection limit is 0.07 zmol which is at least 3 orders of magnitude lower than many other DNA detection schemes. DESIGN OF MOLECULAR BEACON PROBES Molecular beacons are single stranded DNA molecules with a stem-and-loop structure.(Fig.1) The loop portion of the beacon can form a double stranded structure in the presence of its complementary nucleic acid strand. The two ends of the stems of a MB are labeled with a fluorophore and a quencher. INTRODUCTION (a) DNA/RNA analysis plays an extremely important and fundamental role in the rapid development of molecular diagnostics, genetics, and drug discovery. One of the fastest growing areas in DNA/RNA analysis is the development of DNAbased biosensors. A variety of biosensors, both optical and electrochemic al, have been developed for gene sequence analysis and biological pathogen detection[1,2] based on the DNA hybridization technique. In DNA hybridization, the target gene sequence is identified by a DNA probe that can form a double stranded hybrid with its complementary nucleic acid with high efficiency and extremely specificity. For a typical DNA hybridization based biosensor, it requires the steps of immobilization of DNA probes on the sensor surface and washing away the non-specific molecule binding t o ensure specificity. The non-perfect surface modification from immobilization and incomplete washing are the main sources of noise and hence determine the ultimate sensitivity[3,4]. To immobilize probe molecules on the sensor surface and to achieve efficient washing require more fluidic devices to deal with excessive solutions if automation is desired. These are also time and power consuming steps. In addition, the immobilized monolayer could be destroyed by a high temperature condition that limits the post fabrication (chip bonding) choices if a closed sensor is to be developed. All of these issues, which comes from these two cumbersome steps, add complexities to the lab-on-chip design. To remove the immobilization and washing steps, a molecular beacon (MB) based RNA -DNA hybridization technique [5,6,7] was implemented for DNA detection. The molecular beacons are oligonucleotide probes that become fluorescent only upon hybridization with target DNA/RNA molecules. By implementing this technique, the biosensor can still provide high specificity even without washing away the excess non-hybridized probes which are not fluorescent (if they exist in the solution). (b) Hybrid Molecular Fluorophore Beacon Quencher Fig. 1 (a) Before hybridization, the Molecular Beacon remains non-fluorescent because the fluorophore is quenched by the quencher, (b) Molecular Beacon becomes fluorescent after hybridization with targets The sequences of the two stems are five to eight bases long and are complementary to each other. Due to the hybridization of the two stems, the fluorophore and quencher are in close proximity to each other, causing the fluorescence to be quenched by the quencher (Fig. 1(a)). The sequence of the loop, which is typically twenty to thirty bases long, is designed to be complementary to sequence of the target DNA molecules. In the presence of the target DNA molecules, the stronger binding force between the longer loop structure and target DNA will unbind the shorter/weaker stem structures and separate the quencher from the fluorophore(Fig. 1(b)). The sequence of the loop structure is designed according to a portion of the sequence of 16 s rRNA in E.coli (MC41000) and is 22 bases long. The 5' end is labeled with Fluorescein and the 3' end is labeled with Dabycl quencher. The complete sequence is 5' Fluorescein -GCTCG TATTA ACTTT ACTCC CTTCC TCCGA GC - 3’Dabycl. *Patent pending 0-7803-5998-4/01/$10.00 @2001 IEEE Target 431 Metal (2200A) (a) (a) (b) (b) Cr/Au 2200Å Pyrex Glass Glass Si SiO2 5000Å (c) SiO2 (5000Å ) (d) Fig. 4 Anodic bonding of Pyrex and SiO2 with a metal layer partially in between (a)Before bonding, a space of 2200Å between glass and SiO 2 (b)After bonding, the metal is squeezed in between Fig. 2 Cross sections of microchannels bonded with glass (with silicon dioxide and metal layers in between). Sidewalls are coated with a metal layer to make reflection mirrors (a)rhombus channel, (b) v-groove channel (c)trapezoid channel,(d) rectangular channel (a) (b) Au SiO2 Air Water (a) Air (b) Fig. 5 Completely bonded channels (a)Channel is empty (b)Channel partially filled with water and no leak is observed in the Au/SiO2 interface and top Au edges of the channel 100 µm 100 µ m between the channel chip and glass is strong enough to squeeze the metal layer tightly between those two substrates and minimize the clearance in the interface between the non-bonded metal and bonded SiO 2 areas. A complete bonded chip is then injected with water, and no leak is observed along the Au and SiO 2 interface as well as in the squeezed top metallic edges of channel (Fig. 5). (d) (c) 100 µ m 100 µm PMT Fig. 3 Microscopic pictures of channel crosssections (a)rhombus channel, (b) v-groove channel, (c)trapezoid channel, (d) rectangular channel Focusing Lens Band pass filter Laser Input FABRICATION Microchannels with different geometry, and metal coatings on the sidewalls are fabricated to maximize signal-to– noise ratio. Channels with v -groove, trapezoid, and rectangular cross sections were fabricated by KOH and DRIE etching, and channels with rhombus cross sections were made by DRIE pre etching followed by KOH etching (Fig. 2 and 3). The channel width varies from 10µm to 150µm and its depth changes from 20 µm to 100µm. After 5000Å of thermal silicon oxide is grown for electrical isolation, a 2200 Å thin Al or Cr/Au layer is deposited by sputtering or e -beam evaporation. To pattern electrodes and sidewall mirrors in t he deep channels, 10 µm AZP4620 PR is coated, over-exposed and developed to make etching masks or perform lift -off as the case requires. The channel chip is then bonded to a pre -drilled Pyrex glass plate using anodic bonding technique to form a closed channel. In spite of a spacing 2200 Å between the channel chip and the glass plate due to the metallic layer as shown in Fig. 4 (a), the electric field is large enough to pull the two plates together and form a completely bonded channel (Fig. 4(b)). Also even if the Al or Au metal layer does not bond with glass, the bonding strength Beam Expander Band pass filter Beam Splitter Mirror Achromatic Objective DNA sensor chip Fig. 6 Setup of a Laser Induced Fluorescence System INSTRUMENTATION A Laser Induced Fluorescence (LIF) system (Fig. 6) is used for biosensor characterization. An excitation beam (2mW) from an air-cooled Ar ion laser (ILT,100mW) passes into a beam expander (Melles Griot, 09LBZ010) and a band pass filter (Omega, XF1073). It then reflects from a dichroic beam spliter 432 (Omega, XF2037) to a 20 x 0.50 N.A. objective (Rolyn Optics Company, 80.3080), which focuses the beam to a 30 µm spot within the channel. Fluorescence is collected by an objective, passes through the dichroic beam splitter, filtered by a bandpass filter (Omega, XF3003), focused by a focusing lens (Newport, PAC052), and finally collected by a PMT (Hamamatsu, HC12001). The signal from the PMT is transmitted to a data acquisition card and analyzed by a Lab View program. SNR Enhancement (%) 1000.0 865.3 Al Coating 800.0 Au Coating 579.9 566.2 600.0 421.2 400.0 200.0 79.5 34.7 EXPERIMENTAL PROCEDURES 20.6 0.0 The eight hundred bases long nucleic acid targets used for detection were synthesized by polymerase chain reaction (PCR). The sense (5' –CAGAT GGGAT TAGCT AGTAG GTG3') and antisense (5' –GTCTC ACGGT TCCCG AAGGC AC -3') primers derived from the most conserved region of 16s rRNA of 2E.coli.(MC41000) were identical to those used in the previously reported study[7]. Initially, 50 µl DNA solution with concentration of 0.2 µM and 50 µl MB solution of the same concentration were mixed for biosensor characterization. The signal to noise ratio (SNR) and detection limit of channels with various geometries and surface coatings are determined by testing the different channels with the serially diluted solution. Because of the introduction of molecular beacons, two of the major but cumbersome steps, immobilization and washing, for typical DNA hybridization technique were eliminated. This greatly simplified the preparation steps for DNA detection. The mixed solution was pipetted into the inlet of the drilled glass hole on the sensor chip(Fig. 7), and the surface tension force pulls the solution to the coated detection region. A 488 nm light beam form the Ar ion laser was focused onto the coated region, and the existence of target DNA can be identified by checking the intensity of the emitted fluorescence light (λ=512nm) which comes from stretched Fluorescein labeled MB probes. 20 2 0.2 15.0 0.02 Concentration (nM) Fig. 8 SNR enhancement due to Al & Au coating for different sample concentration SNR 25.00 20.00 15.00 10.00 5.00 0.00 Retangular Rhombus Trapzoid V-groove Fig. 9 Comparison of SNR for different channels. The concentration of MB-DNA solution used for testing is 2 nM. Sidewalls in the detection regions of the four channels were coated with an Al layer. The channel width is 80 µm. in the experiment which was 512 nm in air (385 nm in aqueous solution), the average reflectance (normal incidence) for Al thin flim is 0.92, for Au thin film is about 0.40, and for SiO2 thin film is less 0.20 [8]. The magnitude order of refle ctance agrees with the experimental SNR enhancement data. Comparing the SNR of rectangular, V-groove, trapezoid, and rhombus channels, it is found that SNR of the trapezoid channel is 3.0 times higher than that of retangular channel, and is 2.4 times higher than that of rhombus channel (Fig. 9). The experiments are performed by injecting 2 nM sample solution (DNA -MB hybridization products) into the various channels with the same width of 80 µm. It is obvious that channels fabricated by KOH etching have mu ch smoother sidewalls than those made by DRIE etching (Fig.10(a), (b)), and have higher overall reflectivity for the same surface coating condition. For the KOH etched channels, due to the deposition and patterning difficulties in creating a uniform metal coating on the four sidewalls of the rhombus channels(Fig.11), the SNR of the rhombus channels is lower than those of trapezoid and V-groove channels. According the above results, the trapezoid channel is determined to be the optimum design for detection based on the currently employed fabrication techniques. Thus, trapezoid channels coated with Al, Au, and thermal oxide are tested to compare the detection limits. As shown in Fig. 12, the detection limit for a channel with thermal oxide coating is 200 p M while for a channel with Au coating is 20 pM. And for an Al coated channel, Detection channel Reservoir Detection Mirror Electrode Fig.7 A sensor chip with 8 detection channels. RESULTS For the same geometry of microchannels, those with Al coating in the detection region was found to have the highest signal to noise ratio over those coated with Au or SiO 2 in the same detection condition. In addition, micrchannels with Au coating has higher SNR than those without any metallic coating which is SiO 2 surface. The SNR enhancement for the Al and Au coated channels over SiO2 coated channels ranges from 80% to 860% and from 14% to 420% respectively for different concentrations of sample solution as shown in Fig. 8. It is believed that the enhancement is because of the improvement of reflectance which was due to the metallic coatings. In the wavelength range of emitted fluorescence 433 (a) (b) technique, the concentration detection limit is 0.07 zmol with SNR=3 as a threshold. This is approximately three orders of magnitude lower than that for many other DNA detections. It is possible to visualize a single light-emitting molecule but not for DNA detection with high specificity[9,10], thus the 0.07 zmol (about 50 molecules) detection limit is a significant advancement. In the case of a more diluted solution, the metallic mirror can be also used as an electrode to apply positive potential for concentrating negatively charged DNA in order to further improve the performance of the sensor. SiO 2 SiO 2 Al Al Fig. 10 SEM pictures of channels with Al coating (a) KOH etched trapezoid channel, sidewall and bottom are smooth (b) DRIE etched rectangular channel, sidewall and bottom are very rough ACKNOWLEDGMENTS This work is supported by DARPA MTO under a Contract N66001-96-C-83632 managed by SPAWAR. Advice offered by Dr. Jeffery Miller and Mr. Minghsun Liu’s on MB probes and PCR primers are appreciated. Fig. 11 SEM picture of KOH etched rhombus channel with Al coating. The coating is not uniform on the sidewall which affects the SNR of detection. REFERENCES 1. M. Yang, M.E. McGovern, and M. Thompson, "Review Genosensor technology and the detection of interfa cial nucleic and chemistry", Analytica Chimica Acta, 346(1997), 259-275 2. D. Ivnitski, I. Abdel-Hamid, P. Atanasov, and E. Wilkins," Review Biosensors for detection of pathogenic bacteria", Biosensors & Bioelectronics 14 (1999), 599-624 3. Y.F. Chen, J.M. Yang, J.J. Gau, C.M. Ho, and Y.C. Tai.”Microfluidic System for Biological Agent Detection,” The 3rd International conference on the interaction of Art and Fluid Mechanics, Zurich, Switzerland, 2000. 4. J. Gau, E. Lan, et al., Proceedings of the Fourth International Symposium on m-TAS (2000), 509-512 5. X. Liu, W. Farmerie, et al., "Molecular Beacons for DNA Biosensors with Micrometer to Submicrometer Dimensions", Anal. Biochem. 283(2000), 56-63 6. S. K. Poddar, "Detection of adenovirus using PCR and molecular beacon", J. Virological Methods 82 (1999), 19-26 7. T.H Wang, Y. F. Chen, S. Masset, C. M. Ho, Y.C. Tai, "Molecular Beacon Based Micro Biological Detection System," proceedings of METMBS'00, pp295-300 8. M. Bass, “Handbook of Optics”, 2 nd Edition, V.II, Mc-Graw Hill 9. W .P. Ambrose, P.M. Goodwin.,et al. “Single -molecule detection with total internal reflection excitation: comparing signal-to-background and total signals in different geometries.”Cytometry 36(1999), 224-231 10. C. Zander, K.H. Drexhage,” Single -molecule counting and identification in a microcapillary”, Chemical Physics Letters 286(1998), 457-465 Relative Signal Level 30 Al Coating 25 Au Coating SiO2 Coating 20 Back Ground 15 10 5 0 1 -19 7x10 20,000 2 -20 3 -21 7x10 7x10 2,000 200 4 -22 7x10 20 5 -23 7x10 mole 2 pM Fig12 Sensitivity check of channels with different coatings. KOH etched trapezoid channels are used. Channel width and depth are 80 µm and 50µm. The detection limit for Al coated channel is 7x10-23 mole which is about 50 DNA molecules. the detection limit is as low as 2pM which is about only 0.07 zmol (0.07 x 10-21 mole, about 50 DNA molecules) in the 36 pL probe volume (based on a 30 µm dia. focusing spot and 50 µm channel depth). This is approximately three orders of magnitude lower than that for many other DNA detections[1,2]. CONCLUSIONS Inchannel specific DNA detection was achieved by using molecular beacon based DNA hybridization. This process greatly reduces the sample volume required for detection and could facilitate the integration of a biosensor to a µ-TAS. Microchannels coated with metal films with high reflectance are fabricated that increase the signal level in a Laser Induced Fluorescence(LIF) system. By using the side-mirror channel with inchannel sensing 434