Development and characterization of a microheater ...

Development and characterization of a microheater array device for real-time DNA mutation detection Layne Williams1, Murat Okandan2, Alex Chagovetz3, Steve Blair1,3 1 University of Utah, Department of Bioengineering, 2Sandia National Laboratories, 3University of Utah, Department of Electrical and Computer Engineering ABSTRACT DNA analysis, specifically single nucleotide polymorphism (SNP) detection, is becoming increasingly important in rapid diagnostics and disease detection. Temperature is often controlled to help speed reaction rates and perform melting of hybridized oligonucleotides. The difference in melting temperatures, Tm, between wild-type and SNP sequences, respectively, to a given probe oligonucleotide, is indicative of the specificity of the reaction. We have characterized Tm’s in solution and on a solid substrate of three sequences from known mutations associated with Cystic Fibrosis. Taking advantage of Tm differences, a microheater array device was designed to enable individual temperature control of up to 18 specific hybridization events. The device was fabricated at Sandia National Laboratories using surface micromachining techniques. The microheaters have been characterized using an IR camera at Sandia and show individual temperature control with minimal thermal cross talk. Development of the device as a real-time DNA detection platform, including surface chemistry and associated microfluidics, is described. Keywords: DNA hybridization, DNA melting, biosensor, real-time detection, microheater

1 INTRODUCTION DNA analysis is becoming an increasingly important tool in many fields. In addition to genotyping, food testing, forensic investigation, and disease detection have all benefited from the use of DNA testing methods. A penultimate goal of DNA analysis is personalized medicine – tailoring treatment to the specific genome of the patient and even acting to prevent disease. Single nucleotide polymorphisms (SNPs) play a large role in this picture. SNPs are important disease biomarkers and may also indicate significant changes in the behavior of an organism. In humans, for example, SNPs comprise 90% of all genetic variations. Cystic Fibrosis is associated with over 1500 mutations in the Cystic Fibrosis Transmembrane Regulator (CFTR) gene, many of which are SNPs. Most screening for CF is based on up to 32 most commonly observed mutations. For this testing to be expanded, increases in reliability and decreases in cost must be achieved. Miniaturization of testing methods will help to realize these goals. Microscale devices have the advantages over macroscale systems of low power, small sample sizes, rapid heat transfer rates, and increased surface area to volume ratios. Many microscale devices can be batch fabricated to help reduce costs as well. Towards the goal of increasing reliability, we have investigated the use of temperature in SNP detection as a means to increase specificity in rapid DNA analysis. Temperature is often used to increase hybridization rates in an experiment and perform DNA melting. We have also developed a microscale DNA platform with 18 individually controllable heaters for temperature control, providing a miniaturized platform for DNA analysis. The motivation behind the platform design was to use microheaters for local temperature control in DNA detection, and integrate it with an optical waveguide and microfluidics. Others have reported the use of temperature control in microscale DNA analysis systems but these were bulk heaters attached to a microfluidic channel of some sort1-4. Detection methods employed included waveguides and fluorescence microscope systems. A microheater based platform for SNP detection was developed by Kajiyama et al5. They demonstrated individual temperature control on 500 µm square heating zones. However this was not a real-time system and relied on a wash step and then end point scanning. Petersen et al. reported a multi-thermal zone washing system in which a pre-hybridized substrate was washed at various temperatures to determine melting of different oligonucleotide

Microfluidics, BioMEMS, and Medical Microsystems VI, edited by Wanjun Wang, Claude Vauchier, Proc. of SPIE Vol. 6886, 68860K, (2008) · 0277-786X/08/$18 · doi: 10.1117/12.767286

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sequences6. This was also an end point measurement system, as the slide was removed and scanned. The advantage of the system we propose is the incorporation of temperature control with real-time detection, which not only simplifies the necessary steps by eliminating the wash, but provides for data capture during the hybridization and melt experiments.

2 METHODS AND MATERIALS 2.1 Microscale device design The microscale platform was designed to include three components: a planar waveguide for real-time detection, microfluidics for sample delivery, and microheaters for temperature control. The planar waveguide is the substrate for attaching DNA probes. Microfluidic channels overlay the waveguide layer to deliver sample to the surface. Heaters are individually controllable, addressing the specificity of each probe oligonucleotide sequence. Design was performed using AutoCAD with tools specific to the SwIFT-LiteTM processes developed by Sandia National Laboratories7. This process includes low-stress silicon nitride (LSN) layers in place of some of the polysilicon layers normally used in the more familiar SUMMiTTM process. LSN is a capping material which holds up well under sacrificial layer removal by wet etching. LSN became the default choice for the waveguide material, based on the fabrication process for SwIFT-LiteTM. Silicon dioxide (SiO2) which is normally a sacrificial material was instead retained and used as cladding for the waveguide layer. Polysilicon, phosphorus doped to increase its resistivity, serves as the resistive material for the microheaters. Grating couplers were designed to provide input for a light source to the waveguide layer. To keep fabrication costs down the gratings were designed to be made in a batch process with standard photolithography of 0.35 µm feature size. Layer thicknesses were pre-specified and not subject to change, nor were the available materials, see figure 1. To limit thermal cross talk between heating structures and improve control of the device, the heaters were isolated in between layers of silicon nitride and silicon dioxide. The underlying silicon substrate is etched using a Bosch process to help prevent thermal conduction through the device. Microfluidic access was designed to enter from the backside of the device to free the surface for optical interrogation. Microfluidic channels are laid over the top of the device surface, aligned with the heating elements and fluid throughholes. Figure 2 shows the finished design of the device as laid out in AutoCAD.

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Figure 1 -- Cross section of device showing material layers and thickness for the SwIFT-LiteTM process.

2.2 Modeling Modeling of the microheater platform was performed in Comsol Multiphysics. Two models were made, one of an individual heater with resistive elements to determine uniformity, and the other of the entire 18 zone device to show the degree of thermal cross-talk in the substrate and in the microfluidic system. The individual heater contains only one plane of symmetry, so this was used to reduce the model size to half. Boundary conditions were set to insulation on all surfaces except the top and bottom where the interface was with air. The model shows good temperature uniformity on

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the surface of the device. Based on the uniformity of this result, the model of the entire device surface with microfluidics could be simplified by using a constant temperature in for each given heater area and not using the resistive elements. The heaters to be turned on were given a value different from the rest of the surface (30 °C) in the initial conditions. The interface for the surface was set to water and insulation elsewhere. 2.3 Fabrication The microscale platform was fabricated by Sandia as part of the University Alliance program. The device was fabricated in a production run containing multiple devices on a single wafer. As such, the size of the device was that of a single module on the wafer, approximately 2.8 x 6.3 mm. Photolithography masks are generated from the AutoCAD design by an external company. The deposition process is available elsewhere (mems.sandia.gov). In brief, a thermal oxide layer is grown on the silicon substrate to 0.63 µm thickness. Then a 0.8 µm LPCVD LSN layer is deposited. This serves as the base for the rest of the SwIFT-LiteTM process. Structural polysilicon, poly0, is applied and patterned, coated by PECVD SiO2, sacox 1. For this process, the next and final layer was LSN (Figure 1). Once deposition was complete the wafers were sent out for planarization. Upon return to Sandia, the wafers were diced into individual modules. The final processing step was release, or removal of the sacrificial SiO2 layers using an HF/HCl solution. Released devices were packaged in a 40 pin DIP at Sandia. The DIP was laser machined with two holes for backside fluid access. The device fluid access holes were aligned with the package access holes, and sealed around. A gap was left between the bottom of the device and the package for later processing. Figure 3 shows the completed, packaged device including wirebonds. 2.4 Thermal testing The microheaters were tested for uniformity and cross-talk using a thermal imaging system at Sandia. A Quantum Focus Instruments InfraScope 1 camera was used to image a single microheater for high resolution images. The resolution of the thermal camera was 20 µm/pixel with a CCD size of 160 by 120 pixels. Flat black spray paint was applied to ensure imaging of the surface rather than the underlying polysilicon resistive elements. A Keithly 2400 SMU was used to source current and measure the resulting voltage across the resistive elements. Current applied ranged from 0.5 to 3 mA, with resulting voltage ranging from 2.5 to 20 V.

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2.5 Melting Study DNA melting studies were performed in both solution phase (nothing immobilized) and solid phase (probe DNA sequence immobilized). Sequences used were 18 base pairs in length, obtained from the CFTR mutation starting at position 1640, see Table 1. The oligonucleotides were synthesized by Biosearch Technologies. A fluorophore, Quasar 570, was attached on the 3` end of the target sequences. Black Hole Quencher 2 (BHQ2) was attached on the 5` end of the probe sequence, which also contained a 3` amine modification for immobilization. 18mer (1640) wt (target)

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Solution phase melting was performed on a Light Cycler instrument. Samples were loaded in the capillary tubes at concentrations of 300 nM for the probe and 450 nM for target. The temperature range was 37 to 90, stepping in 1 degree intervals. Solid phase melting was performed on quartz microscope slides. Prior to probe immobilization, quartz microscope slides were washed with Alconox, rinsed with DI water, and dried with ultra high purity N2. The slides were then cleaned in O2 plasma for 100 minutes at a pressure of 400 millitorr oxygen and power of 100 W. The slides were transferred to a vacuum oven where glycidoxypropyltrimethoxysilane (GPS) was deposited in vapor phase for 8 hours at 120 °C. The processed slides were then spotted with amine-modified oligonucleotide probes using a single pin noncontact spotter built in-house. The spotting volume was 100 nL, and spots were allowed to dry down while spotting. Spotted slides were stored overnight in a humid chamber at room temperature. After rinsing with cold DI water and drying with N2, the slide was ready for assembly as a microfluidic chamber. A glass slide was used as the cover for the microfluidic chamber. Two holes were drilled in the glass slide for fluid ports and then it received the same surface treatment as the quartz slide prior to spotting. Microfluidic access was achieved using Nanoport connections (Upchurch Scientific Inc., Model N-333). The top of the glass cover was treated with AP8000 adhesion promoter (Dow Chemicals) and then the Nanoports glued in place with DP-190 epoxy (3M). The cover and ports were placed in an oven at 70 °C for 2 hours to cure the epoxy. The chamber was made from 500 µm

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thick VHB adhesive tape (3M, model 4905). The adhesive tape chamber was cut with a sharp blade and placed on the bottom of the cover glass slide, and sandwiched with the spotted quartz slide. The reactive epoxy sites on the quartz slide were blocked with 0.5% BSA. The BSA solution was heated to 50 °C for 1 hour and then 10 mL were pushed through the microfluidic chamber with a syringe pump (KD Scientific, model KDS120), followed by 10 mL of 90 °C DI water. The DI was removed from the chamber and the sample solution loaded into the system. The system placed a vacuum desiccator and degassed for 1 minute to eliminate bubbles in the chamber. The assembled slide was then placed on a temperature controlled mount. The mount uses a Peltier heating device, which was electronically controlled using a feedback circuit and thermocouple. The absolute temperature accuracy was estimated to be 1 °C. The temperature was ramped slowly (10 °C steps every 5 minutes) up to 70 °C to denature the oligonucleotides. Data collection was started at this point and then the temperature dropped quickly to the desired set point, enabling capture of the beginning of the hybridization reaction. A CCD camera (Santa Barbara Instruments Group, ST7XEI) was used to monitor the hybridization. Hybridization of Black Hole Quencher 2 (BHQ2) 5` modified oligos was done at room temperature overnight. A 532 nm laser was endfired into the quartz slide to excite the fluorescence of the immobilized oligonucleotides. The system was then monitored as the Peltier heater was activated. Hybridization was observed by a decrease in fluorescence through quenching of the BHQ2 in a FRET reaction. Melting was observed by an increase in fluorescence.

3 RESULTS Once the devices were received and packaged, testing began with the IR camera. The heaters were designed to have a resistance of 5k
to keep the voltage drop below 50 V for safety requirements while operating. The current applied to the heater was between 1 and 3 mA. An image of four microheaters is shown in figure 4. The image shows very good uniformity across the surface of the heaters, as seen in a plot of the surface temperature in figure.

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The GPS deposition process was characterized on silicon dioxide and silicon nitride chips with water contact angle measurements and XPS. Three cleaning procedures were examined. First RCA cleaning, consisting of a basic etch followed by and acidic etch. This was considered the gold standard method. Chips were rinsed thoroughly with DI and baked for 30 minutes at 90 °C before deposition. Oxygen plasma cleaning was performed to determine the minimum length of time needed to achieve a 0 degree water contact angle on the silicon dioxide and silicon nitride surfaces. At the settings of 100 W and 400 mtorr, the minimum time needed to ensure 0 degrees was 1 minute. This surface was then also rinsed with water and baked for 30 minutes at 90 °C. The final cleaning method examined was oxygen plasma for

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100 minutes. This surface was not rinsed and went straight from the plasma system to the vacuum oven for deposition. The carbon content of both the silicon dioxide and silicon nitride chip surfaces was compared before and after GPS deposition for each of the cleaning methods. The largest change was observed with 100 minute plasma cleaning. Water contact angles obtained from the GPS surfaces for 100 minute plasma cleaning were 62±1 degrees, slightly higher than values obtained for the other cleaning methods. The LSN layer fabricated by Sandia was checked for optical modes with a Metricon prism coupling instrument. Samples with 800 nm LSN and 2000 nm SiO2 were measured. Four TE modes were found in the LSN layer, and refractive index was determined to be 2.188. With optical modes supported, the grating coupler for the waveguide layer was tested on separate chips containing gratings fabricated by Sandia. The gratings on the chips were centered on a rotation stage. A 633 nm laser was used to illuminate the gratings at the calculated coupling angle for the TE0 mode. The surface waveguide was examined to observe any scatter from the guided mode as well as the end of the waveguide. No scatter was observed from the surface nor light exiting the waveguide. Melting studies in liquid phase show the expected result of higher melting temperatures for the given probe with its specific complement target than with a mismatched target. Solid phase melting shows the same trend, however there is a shift in the melting temperature as compared to the liquid phase experiment, see figure 5.

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4 DISCUSSION One of the main advantages of developing this device at Sandia National Laboratories was the use of their surface micromaching processes. They are extremely well characterized and produce remarkable results. With the well characterized fabrication process however came many limitations on the design process. Thicknesses of layers and available materials were predetermined and could not be changed, especially since this device was part of a large wafer containing many devices fabricated with the same process. Still, a well built device that was designed within the fabrication constraints was achievable. The microheaters designed for this device showed excellent uniformity and no observable cross-talk during experiments with the IR camera at Sandia. The IR camera used is a mid-range system, which covers the range from 3 to 5 µm. The silicon nitride and silicon dioxide layers are still transparent in this wavelength range, but the underlying polysilicon was easily imaged. While this gave a measure of the temperature of the polysilicon, the temperature of the silicon nitride surface was the desired measurand. To image this surface the flat black spray paint was applied, in as thin a coat as possible while trying to maintain a uniform surface. It is possible the paint would obscure the thermal measurement, but

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this would only have the effect of decreasing the observed temperature. With the paint on the surface and operating at 3 mA, temperatures in the range of 100 °C were easily achieved which was more than the desired operating temperature of the system. However, characterization of the surface temperature by this method would not be exact. The device was designed to have a thermal mass underlying each heating element. The thermal mass absorbs the heat from the resistive element and then because it is isolated helps to distribute the heat more evenly that the resistive element alone. Briand et al. showed the use of a 10 µm thick thermal mass underlying a heating element helped to achieve uniform surface temperatures in a similar device8. Kajiyama also used a similar thermal mass structure to help isolate individual heaters5. For the Sandia device the thermal mass was made by the use of a “counterbore” etch under each heater to thin the substrate from 400 to 200 µm. Then a second deep etch was done to create the 50 µm wide trench to isolate the thermal mass from the substrate. While this did have the desired effect on the temperature of the surface, the device became very fragile as the 250 µm square and 200 µm thick thermal mass was supported by an approximately 4 µm thick membrane of silicon nitride and silicon dioxide. The packaged device contains adhesives to bond the device in the package, wirebonds from the device to the package frame, and potentially fragile membranes supporting a large thermal mass. With this in mind, the surface chemistry was developed based on dry cleaning methods and vapor phase deposition processes. In the overall assembly process it makes more sense to do surface chemistry after packaging to prevent fouling of the surface in the process. The RCA cleaning method was never intended to be used on the device, but was used as the gold standard for the oxygen plasma processes. Gratings were never fabricated on the devices containing microheaters. This was due to the processing steps required to make the gratings and the fact that this was the only module on the wafer that needed the extra steps. So even though the LSN layer could act as a waveguide according to the data collected from the Metricon, it was not able to be used in the packaged device and prevented the use of the device as a real-time system. A solution to observe hybridization and melting in real-time was developed based on a FRET detection mechanism. The immobilized oligos were labeled with a fluorophore and the target oligos in solution with a quencher. This would prevent labeled oligos in the bulk solution from fluorescing and swamping any signal from the surface while using surface illumination instead of an evanescent field. Experiments on quartz substrates verify the FRET reaction occurs between Quasar 570 and BHQ2. The melting temperature values obtained in liquid phase experiments were slightly higher than the predicted theoretical values shown in Table 1. A possible source of this variation could be evaporation, which would cause an increase in the concentration in solution. It is also not known what effect the fluorophore and quencher have on DNA binding. The melting studies also showed a decrease in the melting temperature of a given double stranded oligonucleotide system in solid phase compared to the liquid phase. One possible cause of this shift could be different concentrations between the two experiments, which were 300 nM probe, 450 nM target in solution and 250 nM target in solid phase assuming all target could bind to probes on the surface. Also a decrease in degrees of freedom for immobilized probe oligonucleotides could be another cause. However, the data collected does support the fact that we can discriminate between the complement and mismatch targets respectively in a melt as there is at least a 5 °C difference between the double stranded systems.

5 CONCLUSIONS Development of the complete device has been a complicated process. Long fabrication times and unexpected variations in the design have necessitated changes in the device assembly and operation. Final assembly of the device is yet to be completed as there are a limited number of packaged devices to work with. Testing of the microheaters has shown proper operation. Surface chemistry and spotting have been developed that are compatible with the packaged device, and preliminary testing has shown the oligonucleotides can be immobilized on a silicon nitride surface. A thinner VHB adhesive, 250 µm as compared to 500 µm, but with the same adhesive properties as was used in the melting studies on quartz substrates will be used to create a channel system on the device, capped off with a cover slip. A pick and place type system has been built to aid in the assembly process. The FRET system has proven successful and will enable realtime detection on the device despite the absence of evanescent excitation from a waveguide. With local temperature

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control and small sample volumes in a microscale device we believe this system will help to realize the goal of more reliable and less expensive DNA analysis systems.

6 REFERENCES 1. H. P. Lehr, M. Reimann, A. Brandenburg, G. Sulz, and H. Klapproth, "Real-time detection of nucleic acid interactions by total internal reflection fluorescence," Analytical Chemistry 75, 2414-2420 (2003) 2. M. Noerholm, H. Bruus, M. H. Jakobsen, P. Telleman, and N. B. Ramsing, "Polymer microfluidic for online monitoring of microarray hybridizations," Lab on a Chip 4, 28-37 (2004) 3. D. I. Stimpson, J. V. Hoijer, W. T. Hsieh, C. Jou, J. Gordon, T. Theriault, R. Gamble, and J. D. Baldeschwieler, "Real-time detection of DNA hybridization and melting on oligonucleotide arrays by using optical wave guides," Proc Natl Acad Sci U S A 92, 6379-6383 (1995). 4. A. Dodge, G. Turcatti, I. Lawrence, N. F. De Rooij, and E. Verpoorte, "A Microfluidic Platform Using Molecular Beacon-Based Temperature Calibration for Thermal Dehybridization of Surface-Bound DNA," Analytical Chemistry 76, 1778-1787 (2004) 5. T. Kajiyama, Y. Miyahara, L. J. Kricka, P. Wilding, D. J. Graves, S. Surrey, and P. Fortina, "Genotyping on a thermal gradient DNA chip," Genome Res 13, 467-475 (2003). 6. J. Petersen, L. Poulsen, S. Petronis, H. Birgens, and M. Dufva, "Use of a multi-thermal washer for DNA microarrays simplifies probe design and gives robust genotyping assays," Nucleic Acids Res 6, 6 (2007). 7. M. Okandan, P. Galambos, S. Mani, and J. Jakubczak, "Development of surface micromachining technologies for microfluidics and BioMEMS," (SPIE-Int. Soc. Opt. Eng, San Francisco, CA, USA, 2001), pp. 133-139. 8. D. Briand, S. Heimgartner, M. A. Gretillat, B. van der Schoot, and N. F. de Rooij, "Thermal optimization of micro-hotplates that have a silicon island," Journal of Micromechanics and Microengineering 12, 971-978 (2002)

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