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Langmuir 2006, 22, 11400-11404
Improved Silicon Nitride Surfaces for Next-Generation Microarrays Jonathan G. Terry,*,† Colin J. Campbell,*,‡,⊥ Alan J. Ross,‡ Andrew D. Livingston,‡ Amy H. Buck,‡ Paul Dickinson,‡ Christopher P. Mountford,§ Stuart A. G. Evans,⊥ Andrew R. Mount,⊥ John S. Beattie,‡ Jason Crain,§ Peter Ghazal,‡ and Anthony J. Walton† Institute for Integrated Micro and Nano Systems, UniVersity of Edinburgh, Scottish Microelectronics Centre, West Mains Road, Edinburgh, EH9 3JF, UK, Scottish Centre for Genomic Technology and Informatics, UniVersity of Edinburgh, The Chancellor’s Building, 49 Little France Crescent, Edinburgh, EH16 4SB, UK, School of Physics, UniVersity of Edinburgh, James Clerk Maxwell Building, Mayfield Road, Edinburgh, EH9 3JZ, UK, and School of Chemistry, UniVersity of Edinburgh, Joseph Black Building, West Mains Road, Edinburgh, EH9 3JJ, UK ReceiVed February 20, 2006. In Final Form: September 22, 2006 This work reports how the use of a standard integrated circuit (IC) fabrication process can improve the potential of silicon nitride layers as substrates for microarray technology. It has been shown that chemical mechanical polishing (CMP) substantially improves the fluorescent intensity of positive control gene and test gene microarray spots on both low-pressure chemical vapor deposition (LPCVD) and plasma-enhanced chemical vapor deposition (PECVD) silicon nitride films, while maintaining a low fluorescent background. This results in the improved discrimination of low expressing genes. The results for the PECVD silicon nitride, which has been previously reported as unsuitable for microarray spotting, are particularly significant for future devices that hope to incorporate microelectronic control and analysis circuitry, due to the film’s use as a final passivating layer.
Introduction The post-genomic era has led to the necessity for analytical tools capable of capturing and processing large quantities of gene expression data. DNA microarrays are a particularly important tool for high throughput transcription studies, as they are capable of parallel measurements of gene expression across the whole human genome.1,2 Currently, DNA microarrays are analyzed using bulky excitation sources, optics, and detectors that do not lend themselves to miniaturization. Recently, there has been growing interest in the study of next-generation devices that incorporate microarray technology with microelectronic circuitry to enable the controlled interrogation of individual probes within an array.3 The use of complementary metal oxide semiconductor (CMOS) technology is particularly attractive in this case, as it allows for the fabrication of large arrays (>106) of addressable sensing elements.4 These arrays are routinely produced to order by silicon foundries and are well suited as substrates for microarray applications. Deposited layers and patterned structures, which, for example, might consist of an array of microelectrodes, can be “postprocessed” directly on top of, and in intimate contact with, underlying CMOS circuitry. A key requirement when fabricating integrated circuits (ICs) is the ability to deposit thin uniform films. One film material of particular importance is silicon nitride, which forms an impervious barrier to diffusion, particularly of moisture and sodium ions. For this reason, it is commonly applied as the final passivation layer in CMOS devices to protect IC circuitry from external * Corresponding authors. E-mail:
[email protected] (J.G.T.);
[email protected] (C.J.C.). † Institute for Integrated Micro and Nano Systems. ‡ Scottish Centre for Genomic Technology and Informatics. § School of Physics. ⊥ School of Chemistry. (1) Heller, M. J. Annu. ReV. Biomed. Eng. 2002, 4, 129-153. (2) Campbell, C. J.; Ghazal, P. J. Appl. Microbiol. 2004, 96, 18-23. (3) Golden, J. P.; Ligler, F.S. Biosens. Bioelectron. 2002, 17, 719-725. (4) Fossum, E. R. IEEE Trans. Electron DeVices 1997, 44, 1689-1698.
contamination that can adversely effect device performance.5,6 This protection is especially important in sensors, where the standard encapsulation of devices proves to be impractical. The process temperature of any material being deposited on top of completed CMOS devices must be such that it does not affect the underlying circuitry. Of particular concern is the interconnect metal, which has historically been aluminum because of its low resistivity and excellent adhesion to SiO2. However, a disadvantage of aluminum is its low melting point (660 °C) and the low Al-Si eutectic temperature (577 °C). This means that, to avoid metal film contamination and restructuring and the significant danger of changing dopant profiles in the CMOS structures, any postprocessing steps should not exceed a temperature on the order of 450 °C.6 In standard IC microfabrication processing, amorphous silicon nitride films are typically deposited by either low-pressure chemical vapor deposition (LPCVD) or plasma-enhanced chemical vapor deposition (PECVD). CVD deposition involves exposing a substrate to one or more gaseous species, which react with (and/or decompose on) the surface to produce the desired coating. LPCVD is popular because the lower pressures tend to reduce unwanted gas-phase reactions and also improve film uniformities. However, temperatures on the order of 800 °C are required to provide sufficient energy for reaction and significant deposition of silicon nitride, which means that LPCVD is only used at the front end of CMOS processes. An alternative to LPCVD is necessary for end-of-line layer deposition after metallization. The PECVD technique has the attraction of reduced process temperatures, as it employs a plasma in the gas to create a flux of free radicals sufficient to produce an acceptable deposition rate at the substrate surface. PECVD silicon nitride depositions are typically carried out at 300-350 °C, suitable for (5) Chang, C. Y.; Sze, S. M. ULSI Technology; McGraw-Hill: New York, 1996. (6) Wolf, S.; Tauber, R. N. Silicon Processing for the VLSI Era: Process Technology; Lattice: Sunset Beach, NC, 1986; Vol. 1
10.1021/la060489v CCC: $33.50 © 2006 American Chemical Society Published on Web 11/03/2006
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deposition of the final passivation layer over CMOS structures. Further details of silicon nitride deposition can be found in refs 6 and 7. In recent years, there has been an increasing interest in the use of silicon nitride films in biosensors and as substrates in biomedical and bioengineering studies.8-12 Modification of LPCVD SiN films has produced surfaces suitable for proteinprotein interactions,10 and PECVD SiN has been used as a substrate for microprotein patterning.11 Recently, LPCVD and PECVD SiN surfaces have been investigated as possible alternatives to glass slides for DNA attachment,12 and it was found that, while LPCVD produced comparable results to glass, PECVD films were less effective. As discussed above, for the close integration of IC technology with, for example, DNA microarray technology, it is critically important that all necessary process steps do not involve temperatures exceeding 450 °C. Consequently, this paper reexamines the use of PECVD silicon nitride films as suitable surfaces for DNA microarray experiments. The objective of the work presented is to investigate the performance of silicon nitride layers in microarray experiments following chemical mechanical polishing (CMP) of the surfaces. CMP is commonly used in IC fabrication to planarize the top surface of wafers, as it is particularly important in maintaining surface topography within the depth of focus of photolithography systems.6 In particular, we were interested in evaluating the performance of PECVD silicon nitride following CMP due to its compatibility as an intermediate layer between attached biomolecules and underlying control and test IC circuitry. Experimental Substrate Fabrication. The silicon nitride films were deposited on 3 in. n-type IC-grade silicon substrates, (100)-orientated with a resistivity of 1-10 Ω cm. Stoichiometric LPCVD silicon nitride films were deposited using a LPCVD furnace tube, using gas flows of 5 sccm dichlorosilane (SiH2Cl2) and 20 sccm ammonia (NH3) with a deposition temperature of 800 °C. The PECVD films were deposited using a parallel-plate PECVD reactor (Surface Technology Systems), which employed gas flows of 40 sccm silane (SiH4) and 55 sccm ammonia (NH3) in a nitrogen (N2) ambient of 1960 sccm at a temperature of 350 °C. The chamber pressure was maintained at 900 mTorr, and the rf power that generated the plasma was set at 20 W with a frequency of 13.56 MHz. The LPCVD and PECVD deposition times were targeted to produce film thicknesses of 200 nm. The actual deposited thicknesses were measured using a Nanometrics Nanospec automatic film thickness system. CMP. The CMP process involves feeding an abrasive, corrosive slurry onto a rotating polishing pad. The wafer is vacuum-held in a rotating chuck and forced onto the pad. The chemicals in the slurry then react with and weaken the material, which is removed by the polishing pad. A number of wafers from each deposition process were planarized. The polishing was performed on a Presi Mecapol 460 polisher using a Klebosol 30H50 colloidal slurry chilled to 10 °C. Each wafer underwent a total of 40 s of slurry-based polishing and a further 90 s of rinsing with 1% (w/v) tetramethylammonium hydroxide (TMAH) solution. The full details of the polishing regime are presented in Table 1. After the wafers had been removed from the polisher, they were rinsed in 10% (w/v) TMAH solution for 10 min followed by a cleaning cycle in an Ultratech mask scrubber to (7) Stoffel, A.; Kovacs, A.; Kronast, W.; Mu¨ller, B. J. Micromech. Microeng. 1996, 6, 1-13. (8) Ilic, B.; Czaplewski, D.; Zalalutdinov, M.; Craighead, H. G.; Neuzil, P.; Campagnolo, C.; Batt, C. J. Vac. Sci. Technol. B 2001, 19, 2825-2828. (9) Gao, H.; Luginbu¨hl, R.; Sigrist, H. Sens. Actuators, B 1997, 38, 38-41. (10) Diao, J.; Ren, D.; Engstrom, J. R.; Lee, K. H. Anal. Biochem. 2005, 343, 322-328. (11) Lee, S.-H.; Lee, C.-S.; Shin, D.-S.; Kim, B.-G.; Lee, Y.-S.; Kim, Y.-K. Sens. Actuators, B 2004, 99, 623-632. (12) Manning, M.; Redmond, G. Langmuir 2005, 21, 395-402.
Langmuir, Vol. 22, No. 26, 2006 11401 Table 1. Process Parameters of the Silicon Nitride Polishing Regime parameter
stage 1
stage 2
stage 3
stage 4
slurry pad rotation (rpm) holder rotation (rpm) back pressure (bar) head pressure (bar) time (s)
30H50 40 40
30H50 25 55 0.5 0.15 30
1% TMAH 40 40 0.25 90
DI water 2 2 0.25 2
0.25 10
remove any slurry particulates that remained on the nitride surfaces. Both polished and unpolished wafers were then diced, using a diamond impregnated saw blade. The resulting 25 × 50 mm slides are more convenient for the spotting technology used in the microarray studies. As a control, a commercially produced amino-silane-coated glass slide from Corning (GAPS II) underwent identical treatment and analysis from this point in the process. Following wafer dicing, surface roughness studies were performed on all the slides, using a Digital Instruments Dimension 5000 atomic force microscope (AFM) in tapping mode, with the values of mean roughness, RA, being obtained from a 5 × 1 µm scan area. The contact angles of static deionized (DI) water droplets on the surfaces were measured three times: (i) immediately following wafer dicing, (ii) after cleaning of the slides, and, finally, (iii) after activation of the surfaces. This was performed using a purpose-built goniometer system, which measured the angle between the surface and a droplet of DI water. The contact angle values were determined for each surface using FTA32 analysis software from First Ten Angstroms. Surface Modification. Cleaning of the silicon nitride microarray samples involved immersion in a mixture of ethanol (420 mL), sodium hydroxide (70 g), and water (280 mL) for 1 h. This was followed by rinsing of the samples four times in DI water and drying by centrifugation at 500 rpm for 1 min in an Eppendorf 5810R centrifuge. Silanization of the silicon nitride slides was performed using a standard procedure.13 In brief, the slides were immersed for 1 h in a solution of amino propyl triethoxy silane (APTES, 1% w/v) dissolved in ethanol/water (95:5) and then rinsed four times in ethanol. The slides were then dried by centrifugation at 500 rpm for 1 min and heated to 100 °C for 10 min. Microarray Fabrication. For the microarray measurements, standard experimental protocols were used to print the microarrays on an Arrayjet Aj100 noncontact inkjet printer. Each array contained a selection of 15 (55mer) oligo probes, chosen for their expression attributes. Each probe was printed 75 times per array in buffer (50% w/v glycerol 0.01% w/v Triton X100), producing spots with an average diameter of 120 µm. Control arrays were printed alongside the wafers on standard Corning GAPSII aminosilane slides, which were treated in the same manner as the silicon nitride slides throughout the experiment. All the slides were then placed on a hotplate at 70 °C for 30 min until dry, after which they were blocked with bovine serum albumen [1% in a buffer of saline sodium citrate (5% w/v), sodium dodecyl sulfate (0.1% w/v)] at 42 °C for 45 min. The slides were finally rinsed in DI water followed by 2-propanol and centrifuged dry at 500 rpm for 2 min. Microarray Experiment. Target RNA was extracted from bone marrow-derived macrophage (BMMφ) cultures infected with murine cytomegalovirus (MCMV). Cultures were prepared from 10-12week-old BALB/c mice, which were sacrificed, and their femurs were lavaged to provide a progenitor stem cell population. Progenitor stem cells were seeded in six-well plates at a density of 8 × 105 in Dulbecco’s modified Eagle’s medium (DMEM)/F-12 (1:1) + Glutamax I (Gibco catalog no. 31331-028) supplemented with 10% heat-inactivated fetal calf serum, 10% conditioned L929 media, 10 000 units/mL of penicillin G sodium and 10 000 µg/mL of streptomycin sulfate (Gibco catalog no. 15140-122). Cultures were maintained for 7 days, refeeding on day three and day five. Cultures were then infected at a multiplicity of infection equal to 1 (moi ) 1) with MCMV on day 7. All infection inoculums were made up (13) Laureyn, W.; Nelis, D.; Van Gerwen, P.; Baert, K.; Hermans, L.; Magnee, R.; Pireaux, J.-J.; Maes, G. Sens. Acuators, B 2000, 68, 360-370.
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Table 2. Film Thickness and Surface Roughness Values for the Silicon Nitride Surfaces and Starting Silicon Substratesa layer
thickness (nm)
roughness (nm)
LPCVD SiN LPCVD SiN (cmp) PECVD SiN PECVD SiN (cmp) bare silicon wafer
233.7 208.8 205.5 164.6
0.228 0.114 0.226 0.095 0.092
a
The term cmp refers to those samples that have been polished.
Table 3. Contact Angle Measurements from the Silicon Nitride Surfaces and a Commercial Glass Slide Following Processing, Cleaning and Silanizationa slide
as-processed
cleaned
silanized
LPCVD SiN LPCVD SiN (cmp) PECVD SiN PECVD SiN (cmp) commercial GAPS
28.8 ( 0.2 30.7 ( 0.1 26.3 ( 0.1 37.7 ( 0.1
9.8 ( 0.4 19.9 ( 0.3 8.0 ( 0.3 4.8 ( 1.3
58.5 ( 0.4 53.5 ( 0.5 59.1 ( 0.7 59.1 ( 0.7 41.2 ( 0.9
a
in DMEM/F-12 (1:1) + Glutamax I supplemented with 2% heatinactivated fetal calf serum, 10% conditioned L929 media, penicillin, and streptomycin. Inoculums were left to absorb onto monolayers for 1 h and removed, then the monolayers were washed in phosphatebuffered saline and the maintenance media was renewed. Target RNA was obtained by lysing BMMφ cultures with Trizol as per the manufacturers instructions. Lysates were stored at -80 °C and RNA extraction completed within one week. The air-dried RNA pellet was resuspended in RNAase free H2O and stored at -80 °C. RNA quantification was carried out using a Thermospectronic Biomate 5 spectrophotometer; the A260:A280 was also used to determine RNA purity. An Agilent Bioanalyzer system was used to further assess RNA quality. Samples were run using an RNA 6000 Nano Assay (Agilent Technologies) to detect the presence of two ribosomal peaks, 18S and 28S, indicating good RNA integrity. RNA labeling was carried out using the Agilent low RNA input fluorescent linear amplification method. This kit (Agilent catalog no. 51843523) was used to generate Cyanine5 (cy5) fluorescently labeled complementary RNA (cRNA) following the manufacturer’s instructions. The RNA sample was cleaned using a Qiagen Rneasy purification column. Hybridization. The arrays spotted onto each of the slides were hybridized with 5 µg of cy5-labeled cRNA and placed in a fast slide hybridization hybe chamber (Camlab) for 16 h at 60 °C. The slides were washed in 1 X SSPE (sodium chloride-sodium phosphateEDTA buffer, Amresco 0810)/0.5% N-Lauroylsarcosine (Sigma L-7414), dipped in 2-propanol, and then spun dry at 500 rpm for 2 min before scanning. Scanning and Analysis. The wafers were scanned on a Packard Scannarray 5000 five times at full laser power with varying PMT from 100 to 60%. Scanned images were quantified using Quantarray v.3 (Packard Biochip Technologies). The best scan was selected on the basis of the saturation of pixels and linear range.
Results & Discussion Substrate Characterization. Measurements were taken across each wafer using a film thickness monitor, and the averaged data is presented in Table 2. The LPCVD silicon nitride deposition time was 150 min (rate: 1.56 nm/min), while the PECVD films were deposited in 10 min (rate: 20.6 nm/min). The same polishing regime removed 24.9 nm of LPCVD nitride at a rate of 37.4 nm/min compared with 40.9 nm of PECVD nitride, a rate of 61.4 nm/min. Silicon nitride films deposited by PECVD are nonstoichiometric and can contain a considerable amount of hydrogen, resulting in a film that is effectively SixNyHz.6,7 This high hydrogen content produces a low-density film that experiences higher etch rates, which explains the faster removal of material observed during the CMP process. The mean roughness measurements made using the AFM are also presented in Table 2. As expected, polishing the silicon nitride films resulted in a reduction in the mean roughness values of both LPCVD and PECVD samples. These values are very close to the practical minimum roughness, that is, the roughness of the underlying bare silicon wafer, measured at 0.092 nm. The larger post-CMP value of LPCVD compared to PECVD may be due to greater oxidation of the LPCVD surface during polishing,
All values are given in degrees (°).
causing a slight roughening of the surface.14 However, the difference is minimal. Table 3 presents the contact angles of DI water droplets on each of the silicon nitride surfaces. Three measurements were made: (i) immediately following dicing of the completed substrates, (ii) after the slides underwent a standard cleaning procedure, and (iii) after the surfaces had been activated using the APTES silanization process. The measurement system takes a brief movie of the droplet after placement on the surface in the center of each sample (the area spotted during the microarray experiments), and the software then calculates the contact angle for each frame. The results given in Table 3 are an average of 20 frames of each surface. The as-processed values of 26-28°, those of the freshly diced wafers, agree with those in previously published work.10 The effect of polishing is to decrease the hydrophilicity of the surfaces, shown by the increase in the contact angle. This is in accordance with Wenzel’s law, where hydrophobicity (for angles > 90°) or hydrophilicity (for angles < 90°) is enhanced by an increase in roughness.15 The greatest change in contact angle is seen with the PECVD surfaces, which is in line with the greater reduction in surface roughness on these samples, achieved by the CMP process. After the slides are cleaned, the surfaces are considerably more hydrophilic with contact angles as low as 4.8 ( 1.3° (with the comparatively large error in this particular measurement being due to the difficulty of the software to automatically determine such a small contact angle). Again, these values are in agreement with previous studies and are consistent with a high density of hydroxyl groups at the interface of the film and the droplet.10,12 The final measurements were made immediately after the surfaces had been silanized and show a marked increase in the contact angle values, and hence the hydrophobicity of the surfaces, again in agreement with previous work.10,12 Wang and Jin attribute this increase to the long aliphatic chains of APTES, which produce an increase in hydrophobicity over that seen on hydroxyl surfaces.16 Microarray Results. To assess the differences between different SiN surfaces as DNA microarray substrates, we spotted well-characterized probes corresponding to genes in a wellcharacterized system (mouse macrophage cells infected with mouse cytomegalovirus) and compared the absolute fluorescence values obtained for each set of probe replicates. This allowed us to compare absolute amounts of labeled RNA binding to each slide. Figure 1 shows representative microarray spots, after hybridization, on each type of array slide used. It can be seen, in contrast to previous reports,12 that, in all cases, the overall spot morphology and uniformity were of a consistently high standard. There are no instances of common artifacts such as “comet tails” or “coffee-cup rings”, which are complicating factors in data (14) Yang, G.-R.; Zhao, Y.-P.; Hu, Y. Z.; Chow, T. P.; Gutmann, R. J. Thin Solid Films 1998, 333, 219-223. (15) Berthier, J.; Silberzan, P. Microfluidics for Biotechnology; Artech House: Norwood, MA, 2005. (16) Wang, Z.-H.; Jin, G. J. Immunol. Methods 2004, 285, 237-243.
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Langmuir, Vol. 22, No. 26, 2006 11403 Table 4. Fluorescence Fold Changes of Microarray Spots as a Result of CMP and p Values from a Comparison of Data from Unpolished and Polished Samples p value from ratio of p value from ratio of fluorescence t-test comparing fluorescence t-test comparing of polished/ polished with of polished/ polished with unpolished unpolished unpolished unpolished PECVD PECVD LPCVD LPCVD
Figure 1. Images of the morphologies of post-hybridization microarray spots on silicon nitride slides with a commercial aminecoated glass slide for comparison. All were collected using identical scanner settings. Each image shows a positive control spot (left) and a spot corresponding to the gene of interest (right).
Figure 2. Fluorescence intensity of microarray spots corresponding to (a) negative control genes (1-5 from left to right), (b) positive control genes (1-5 from left to right), and (c) test genes (1-5 from left to right) on glass and SiN wafers, bars show standard errors (n ) 75).
analysis. We attribute this to a combination of the extremely uniform spot deposition process and the quality of both the underlying substrate and the surface modifications carried out. Figure 2 shows the microarray results obtained following hybridization of the probes with a mixture of RNA targets that were labeled with cy5 dye. Duplicate slides with 75 replicates of each probe on the four distinct silicon nitride films, alongside a market-leading coated glass slide (Corning GAPSII), were examined, and target binding performance was evaluated by comparison of spots corresponding to positive control genes,
neg 1 neg 2 neg 3 neg 4 neg 5 test 1 test 2 test 3 test 4 pos 1 pos 2 pos 3 pos 4 pos 5
1.59 1.68 1.42 1.54 1.64 2.17 2.34 2.30 3.74 2.69 3.21 1.94 2.86 2.67
1.76 × 10-07 8.51 × 10-07 1.11 × 10-22 5.94 × 10-21 3.32 × 10-09 6.80 × 10-11 5.97 × 10-05 3.19 × 10-41 6.35 × 10-48 6.39 × 10-74 9.15 × 10-40 2.51 × 10-64 1.72 × 10-51 7.80 × 10-29
2.09 1.65 0.91 2.09 2.11 2.66 1.99 3.72 2.85 4.54 3.22 5.29 2.99 4.03
1.19 × 10-21 7.17 × 10-20 6.48 × 10-30 2.34 × 10-4 2.65 × 10-22 4.86 × 10-33 3.65 × 10-09 2.20 × 10-26 2.43 × 10-24 2.01 × 10-38 9.04 × 10-29 3.16 × 10-43 5.23 × 10-38 1.16 × 10-27
negative control genes, and sample genes of interest relating to viral gene expression. To allow a comparison of absolute fluorescence from all the slides, they were scanned using the same PMT setting. To achieve this, the microarray scanner settings were chosen to ensure a minimum of saturated spots, while maintaining the highest possible fluorescence signal. This allows the highest possible linear range of detection. The results show that, in all cases, the fluorescence from negative control spots (Figure 2a) is low, indicating that none of the surfaces investigated exhibited particularly high levels of nonspecific binding of labeled RNA. There is a slight but statistically significant rise (shown in Table 4) in the mean fluorescence from negative control spots on both LPCVD and PECVD samples after polishing (P values from a t-test < 0.05, corresponding to 95% confidence in all cases). This may result either from the improved binding of probe DNA after polishing, leading to higher levels of nonspecific DNA-RNA binding, or from increased nonspecific binding of RNA to the polished surface. The positive controls span a range of gene expression levels as can be seen from Figure 2b. Significantly, the pattern of expression is the same across the SiN slides. In the case of GAPSII (the market-leading, gold standard surface), the signal for positive control 3 is so strong that the detector is saturated (detection limit 64000). Although this makes the pattern of gene expression appear different to the SiN slides, the other positive controls broadly agree. In all cases, the signal from positive control genes is significantly higher than any of the negative control genes on the same surface (p < 0.05, not shown). On average, in the case of LPCVD SiN, the fluorescence from positive control spots shows a 2.7-fold increase upon polishing (Table 4). On PECVD SiN, the positive control spots show a 4.0-fold increase in fluorescence upon polishing. Like the positive controls, the test genes show a range of expression levels, from relatively high for test gene 4 to relatively low for test gene 5. Importantly, they show the same pattern of expression across the different SiN surfaces. Similarly to the positive controls, polishing causes, on average, a 2.6-fold increase in the fluorescence from test gene spots on LPCVD SiN. On PECVD SiN, polishing causes a 2.8-fold increase in the average intensity of test genes spots. This is particularly important in the case of PECVD SiN since it allows a real improvement in the discrimination of gene expression. Prior to polishing, there is a relatively small difference between the fluorescence from test
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gene 5 and either negative control 3 or 5 (p ) 0.06 and 0.79 respectively). However, the polishing of PECVD SiN allows discrimination of a statistically significant difference between the test gene 5 and negatives controls 3 and 5 (p ) 2.8 × 10-6 and 1 × 10-4 respectively). This demonstrates that the higher fluorescence from test gene spots on polished surfaces allows for a greater discrimination of low levels of gene expression, even though there is also a slight increase in fluorescence from the negative control spots.
Conclusion We have shown that chemical mechanical planarization/ polishing (CMP) of LPCVD and PECVD silicon nitride films results in a increase in fluorescence from the specific binding of cy5-labeled RNA. This makes these improved substrates for microarrays by giving a larger window of discrimination of gene expression and allowing detection of low expressing genes. This is particularly significant for the films deposited by PECVD, as
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the low process temperatures involved make them a compatible technology for integration with CMOS circuitry. Therefore, this is a significant result for future applications that require integration of biomolecular surface attachment with underlying control and sensing microelectronics. Acknowledgment. We would like to thank Andrew Brooke and Andrew Bunting for their assistance and advice with elements of the IC fabrication. This work was carried out as part of a UK Department of Trade and Industry Beacon Project within the Harnessing Genomics program, and we gratefully acknowledge their financial support. Supporting Information Available: Probe sequences for microarray fabrication. This material is available free of charge via the Internet at http://pubs.acs.org. LA060489V
