Fabrication Technology of Periodical Nano-cavities for Bio-analytical Applications

Transcription

1 Fabrication Technology of Periodical Nano-cavities for Bio-analytical Applications O.M. Piciu 1, M.C. van der Krogt 1, M.W. Docter 2, P.M. Sarro 1, A. Bossche 1 1 Delft University of Technology DIMES/EWI, The Netherlands Mekelweg 4, 2628 CD, Delft, o.piciu@ewi.tudelf.nl 2 Delft University of Technology, Department of Imaging Science and Technology, Delft, The Netherlands Summary: In this paper we present a process technology based on electron-beam lithography and lift-off technique, for fabrication of periodical nano-cavities in thin metal layers on glass substrates. Every cavity serves as a reaction chamber for molecular analysis (DNA hybridization, protein immuno-assay). The phenomenon that stands for the bio-detection is represented by the recently discovered optical effects that appear at the passing light through periodically distributed sub-wavelength apertures in optically thick metal films, i.e. spectral selection, enhanced transmission and small optical diffraction [1]. Keywords: periodical nano-cavities, electron-beam lithography, enhanced light transmission I. Introduction In the last years an important part of the research work done all over the world in technology has been directed toward devices for bio-chemical analyses. The specific market needs low cost test tools that use reduced amount of reagents and samples with high reaction speeds and best various parameters detection. When using the optical method for detection, many times the miniaturization of the system has a big limit in the resolution. The recently discovered optical effect that appears at the transmission of the light through sub-wavelength apertures, allow us to develop a novel bio-analytical device. The atto-liter titer plate for high-speed molecular analyses represents our current point of interest. The basis of the device is an optical nano-hole array in a thin metal film (i.e. 200nm gold), where each hole serves as a reaction chamber. The holes are first filled (individually or as sub-arrays) with a solution containing certain molecules necessary for detection (DNA sequences, antibodies, antigens). These molecules bind to the gold substrate through a sulphur-gold chemical bond. In the next step, a transparent plate with embedded nano-channels covers the holes. Via these channels, the sample containing the target molecules is led over the holes. A chemical

2 recognition between the target molecules and the molecules bound to the gold substrate takes place. The detection can be made in two different ways: Excited fluorochrome-bound molecules inside the holes will be visible as transmitted light; Gathered molecules inside the holes can change the optical properties of the transmitted light. Therefore, the device is a titer plate with up to several millions of reaction wells per square centimeter of chip area, instead of 20 wells per square centimeter of chip area as recently developed nano-liter titer plates usually have, the method allowing one to make a detection through ~10^4 holes in parallel, receiving a large amount of information simultaneously. Another advantage is the use of different molecules in every hole (or in every square unit) in order to have parallel, real time analyses in a single experiment. Dispensing methods for this purpose are in development at the moment, but of second interest. II. Technology The proposed method in this paper is a lift-off technique. Fused silica samples (19x19 mm) were first ultrasonically cleaned in HNO3 100%, DI (de-mineralized) water and IPA (isopropyl alcohol), each time for 2 minutes, and finally spin-dried. Next, a 20nm thick chromium film was sputtered on top of the substrate (using an Alliance Concept Ac450 machine and a deposition rate of ~20nm/min). This chromium layer serves as a reflective layer to facilitate the focusing of the Electron Beam Pattern Generator (EBPG) and is also used as a conductive layer for electron discharging during the electron beam (e-beam) exposure. On top of the chromium, a bi-layer resist scheme was applied. The bottom layer is HPR-504 (from Olin) which is an organic, positive tone resist for NUV (near ultraviolet) lithography. The top layer is hydrogen silsesquioxane (HSQ=FOx-12 from Dow Corning), as previously demonstrated inorganic, negative tone resist for highresolution electron beam lithography [2] [3]. First the photo-resist was spin-coated at 5000 rpm for 55 seconds on a Fairchild spinner, after using a primer (HMDS - hexamethyldisilazane) to enhance its adhesion to the metal. Then, it was hard baked at 100ºC, 200ºC and 250ºC, each for 2 minutes, and then over-coated with the HSQ resist, at 6000 rpm for 45 seconds, and baked at 150ºC and 220ºC, again for 2 minutes each. The e-beam resist had a thickness of~150nm and the total thickness of the bi-layer was ~1.1µm. Furthermore, different exposure tests were realized using a Leica EBPG 5000+, in order to find out the correct dose and to overcome issues as proximity effects and overexposure. Finally, using an acceleration voltage of 100 kv and an aperture of 400µm, the following settings were determined: with a beam step size (BSS) of 5nm and an estimated spot size of 8nm, a dose of 4400µC/cm2 for 100nm structures, and a dose of 2400µC/cm2 for 150nm structures; square-dots with periodical and respective

3 random distribution were patterned into the e-beam sensitive resist. After the exposure, the development was done in Microposit MF322 pure solution (Shipley), for one minute, and then the samples were rinsed in DI water and spin-dried. The next process step was to transfer the dot-array-pattern, through reactive ion etching (RIE) pillars into the hard baked HPR-504 photo-resist, using O2 plasma, with a Leybold-Heraeus Z401S parallel plate type RIE reactor. The etching rate of the resist was ~30nm/min at 20sccm O2, 20W, 1.4µbar, and the process was real-time controlled via an interferometer (Sofie Instruments). After obtaining the pillars, each sample was electron-gun evaporated with 200nm of gold (Au), using a Leybold-Heraeus L560 evaporator. In the first tests, the gold was deposited at an evaporation rate of 5Ǻ/s, but due to the fact that these conditions introduced small gold spheres on the surface, which made it too rough for our application, we decreased the evaporation rate to 1Ǻ/s, obtaining a much smoother surface. In the last step of the process, the pillars were lifted-off in fuming HNO3 at 40ºC for 6 minutes, followed by an ultrasonic cleaning for 3 minutes in the same conditions. Finally, the samples were rinsed with IPA (isopropyl alcohol) and spin-dried. Inspection and pictures were realized with a Philips XL30SFEG scanning electron microscope (SEM). III. Results and discussions With the above presented technique, different samples have been obtained: square-hole arrays in gold with the hole size of 150nm or 160nm and the pitch of 750nm or 1050nm (see Figure 1 b)) randomly distributed square-holes in gold with the same hole size as the previous sample and with the same filling fractions (see Figure 2 b)) The arrays are intended to be integrated into the atto liter titer plate device, as reaction chambers where the optical detection takes place. Therefore, to optically characterize the nano-hole arrays we measured the intensity of the light through both periodically and randomly distributed structures, using Koehler illumination, with an upright optical microscope (Leica DM-RXA). Furthermore, we calculated the transmission by dividing the obtained spectra, corrected for the background light, with the spectrum of the light source, and plotted the results as can be seen in the figures below. The measurements were done in the spectral range of 400nm and 800nm, where the sensitivity of the system is optimal.

4 corrected spectra Au a) I_measured/I_illumination (arb. units) wavelength [nm] b) c) Figure 1. a) Spectra of the transmitted light through the periodically distributed square-holes in Au, corrected for the background light represents the array with the hole size of 150nm and 750nm pitch, 0407: hole size of 150nm and 1050nm pitch, 0408: hole size of 160nm and 750nm pitch, 0409: hole size of 160nm and 1050nm pitch; b) SEM picture of a square-hole array in 200nm of Au, with the hole size of ~160nm and pitch of 750nm; c) CCD image of the light through the nano-hole array, hole size 160nm and 1050nm pitch.

5 corrected spectra AuR a) I_measured/I_illumination (arb units) wavelength [nm] b) c) Figure 2. a) Spectra of the transmitted light through the random distributed square-holes in Au, with the same filling fraction as the square-hole arrays, corrected for the background light represents the area with the hole size of 160nm, 0403: hole size of 120nm, 0404: hole size of 150nm; b) SEM picture of random distributed square-holes in 200nm of Au, with the hole size of ~150nm; c) CCD image of the light through the random holes, with the holes size of 160nm. Enhanced transmission, small angular diffraction and spectral selection of light passing through an array of sub-wavelength apertures made in optically thick metal films, have been identified as extraordinary light properties. Comparing the two spectra of the transmitted light through our randomly and periodically distributed holes in gold on glass, when using the same filling fraction (the same number of holes of the same size /

6 same unit area), it came out that the intensity of the light through the periodically distributed holes is more than of a factor of seven higher than through random holes distribution. Furthermore, the spectrum of the arrays in gold shows a series of different peaks at wavelengths between 550nm and 750nm, proving the coupling of the surface plasmons with the photons. The peaks are still broad, as Koehler illumination has been used for the measurements. More defined peaks might be obtain using collimated light. IV. Conclusion Different nano-hole arrays with the hole size between 100nm and 200 nm have been fabricated in gold on glass, using negative tone electron beam resist, electron beam lithography and lift-off technique. Furthermore, for the optical characterization of the arrays, randomly distributed holes in the same substrates have been fabricated, using the same filling fractions and the same technique. An enhanced transmission of light has been observed for the periodically distributed holes versus the random holes distribution. Due to their higher and uniform intensity, the arrays are suitable to be further integrated into the atto-liter titer plate device. Acknowledgment This work was financially supported by the Stichting voor de Technische Wetenschappen (STW) and was conducted in the Nanofacility Department of the Kavli Institute of NanoScience, TU Delft, The Netherlands. References [1] T.W. Ebbesen, H. L. Lezec, H. F. Ghaemi, T. Thio, P. A. Wolff Extraordinary optical transmission through sub-wavelength hole arrays, Nature, 391, , 1998 [2] M. J. Word, I. Adesida, P.R. Berger, Nanometer-period gratings in hydrogen silsesquioxane fabricated by electron beam lithography, J. Vac. Sci. Technol. B 21(6), Nov Dec 2003 [3] F. C.M.J.M. van Delft, J. P. Weterings, A. K. van Langen-Suurling, H. Romijn, Hydrogen silesquioxane/novolak bilayer resist for high aspect ratio nanoscale e-beam lithography, J. Vac. Sci. Technol. B 18(6), Nov/Dec 2000