- Email: [email protected]
Silicon thin-film UV filter for NADH fluorescence analysis
Sensors and Actuators A 97±98 (2002) 161±166
Silicon thin-®lm UV ®lter for NADH ¯uorescence analysis V.P. Iordanova,*, G.W. Lubkinga, R. Ishiharab, R.F. Wolffenbuttela, P.M. Sarrob, M.J. Vellekoopc a
Electronic Instrumentation Laboratory, Delft University of Technology, DIMES, Mekelweg 4, 2628 Delft, The Netherlands Electronic Components, Technology and Materials Laboratory, Delft University of Technology, DIMES, Delft, The Netherlands c Institute of Industrial Electronics and Material Science, Vienna University of Technology, Vienna, Austria
b
Received 1 June 2001; received in revised form 2 November 2001; accepted 14 November 2001
Abstract A new optical detector system for ¯uorescence analysis in on-chip high-speed screening arrays is described. It consists of a photodiode covered by a re-crystallized silicon ®lm, which acts as a single-®lm optical ®lter. The crystalline structure of the silicon ®lm has a strong in¯uence on the optical properties of the ®lter. The fabrication process and characterization of a polycrystalline Si thin-®lm on glass are reported. The method is compatible with standard CMOS processes. The optical properties of the designed ®lter are such that ¯uorescent light of wavelengths of 420 nm or higher can be measured in the presence of (UV) excitation light (340±360 nm). A selectivity exceeding 35 dB has been demonstrated. The technique has the potential for 50 dB selectivity and higher. NADH concentration measurements in the range 0±1 mMol/l illustrate the application of the system for determination of enzymatic activity of different analytes. # 2002 Elsevier Science B.V. All rights reserved. Keywords: UV ®lter; Fluorescence; Biosensor; High-speed screening
1. Introduction High-speed screening (HSS) on arrays implemented in silicon substrates is one of the new tools investigated for biochemical analysis. Such devices consist of an array of subnanoliter wells in which chemical or biochemical samples are examined. By carrying out assays in small volumes [1±3], signi®cant savings can be achieved in the cost of targets, compounds and reagents. The miniaturization of an assay screen also allows more measurements to be carried out in parallel in the same time. Such subnanoliter reactor arrays use one million times less sample than the commonly used 96-reactor titer plates. Fluorescence is an attractive method of detection in assay analyses. It has been used for monitoring of different enzymatic reactions and for a variety of compounds including, but not limited to, pesticides, vitamins and amino acids [4]. Fluorescence detection is a ``low background'' technique, as opposed to absorbance. In an absorbance detector, the measured signal is related to the difference in light intensity with a sample present versus the signal in the absence of a sample. For lower levels of analyte, this difference becomes
* Corresponding author. Tel.: 31-15-278-3342; fax: 31-15-278-5755. E-mail address: [email protected] (V.P. Iordanov).
increasingly smaller, and therefore increasingly harder to detect. The ¯uorescence detector, however, measures light emitted from the solvent in an otherwise dark background. The result is a much lower detection limit, limited by the electronic noise and the dark current of the detector. The other primary advantage of ¯uorescence is selectivity. The two major reasons for greater selectivity, as opposed to absorbance detection are: (1) most organic molecules absorb ultraviolet (UV)/visible light but, not all ¯uoresce; (2) the wavelength of excitation light is in a different spectral range, compared to the emission spectrum. Many ¯uorescence detectors are commercially available today. Each has its own instrumental variables, and is optimized for a certain application. In our silicon subnanoliter array, the wells can be read out in parallel by using a lens and camera [5], but this method is bulky and expensive. An attractive solution is to equip each well with its own photodetector. Using monolithic integration for fabricating the array makes it possible to do the screening electronically. This makes higher speed scanning possible in comparison with mechanical screening. A variety of enzyme reactions can be monitored using the conversion of NAD (nicotinamide adenine dinucleotide) to its ¯uorescent product NADH. We are directing our efforts towards designing a sensor array for NADH concentration measurements in each well. NADH absorbs light of wavelength 340 nm and radiates secondary (¯uorescence)
0924-4247/02/$ ± see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 4 - 4 2 4 7 ( 0 1 ) 0 0 8 4 8 - 2
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Fig. 1. Schematic cross-view of a subnanoliter reactor including a photodetector.
photons with a wavelength of 450 nm. For ¯uorescence detection each reactor cell contains a photo-sensor at the bottom. A possible method to illuminate the analyte is by irradiating the reactor chambers from the side by means of optical waveguides. Such a con®guration is attractive for a single chamber reactor, but in the case of an array a complicated network of waveguides is required in order to ensure that every well receives an equal amount of primary light. A con®guration where the sensor array is illuminated from above (Fig. 1) is the simplest from a technological point of view and allows the use of very simple optical equipment. However, a large fraction of the primary light will pass through the analyte and reach the detector. This light will cause a strong background signal. Therefore, an optical ®lter is required in order to suppress the undesired primary light. Although it is possible to place the junction of the photodiode such that it is sensitive to a selected spectral range only, this would not be suf®cient to provide the required selectivity [6]. As discussed in the text below, silicon appears to exhibit the properties needed to form a UV ®lter [7,8]. Furthermore, a Si-based ®lter structure can be implemented using existing chip processing technology. 2. Optical absorption in Si The absorption process for photons with energy near the bandgap is strongest in direct bandgap semiconductors since the photon can directly cause a transition of an electron from the valence band to the conduction band. When a semiconductor does not have a direct bandgap, absorption of a photon only takes place if a phonon (or lattice vibration) participates in the process. The absorption coef®cient of photons in crystalline Si, an indirect semiconductor, is lower than that of a direct material
Fig. 2. Absorption coefficient of silicon.
and rises only slowly at energy levels near the bandgap (1.12 eV). Once the photon energy is suf®cient to cross the direct gap region (3.4 eV), the absorption coef®cient increases rapidly since direct transitions are possible. Apart from phonon scattering there are also other scattering mechanisms such as alloy scattering and impurity scattering, which can cause optical absorption in indirect semiconductors. As a result, ``poor'' quality indirect semiconductors have a higher absorption coef®cient than pure indirect materials (Fig. 2). In amorphous Si (a-Si), e.g. the absorption coef®cient is quite strong and its absorption properties resemble those of a material with a direct bandgap of 1.12 eV [9]. 3. Laser annealing of a-Si layer Poly-Si ®lms were formed on glass by irradiation of excimer-laser light on an a-Si ®lm with thickness of 75 mm. Excimer-laser basically uses emission of deep UV light from excited ``dimmer'' of rare gas halide, such as XeCl, XeF and ArF. The excimer-laser light has unique characteristics such as a short-wavelength ranged from 150 to 350 nm, a short pulse width of a few tenths of nanosecond, and high output power of about 1 J. Since Si has a strong absorption of the deep UV light, the absorption is limited to a very thin layer of a few tenths of nanometer, where heat is generated by lattice vibration. Since the pulse duration is very short, the penetration of the heat in the substrate is very shallow. The excimer-laser has been used for melting of thin a-Si layer on substrate, such as glass, and to transform it into (poly)crystalline Si [10]. The typically obtained grain size of the excimer-laser crystallized poly-Si is about 100 nm. The grain size takes the maximum value of about 1 mm with the laser energy which Si ®lm is almost completely melted with [11]. By applying
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penetration of the undesired primary (UV) light, while permitting the passage of the secondary (¯uorescence) light. The optical properties of thin-®lm layers (transmittance and absorption) were simulated with TFCalcÐThin-Film Design Software, Version 2.9, Software Spectra. In order to verify the results, the optical transmittance of amorphous and crystalline silicon was measured. The measurements are made with the setup shown in Fig. 4 using a calibrated Hamamatsu S1226 diode. An additional measurement in the UV range was performed using an argon laser, Spectra Physics, Model 2020-05, l 365 nm, optical power 240 mW cm 2. 5. Filter experiments
Fig. 3. XeCl excimer-laser setup for a-Si re-crystallization.
multi-shot irradiation, the substantial grain growth is induced, which yields to grain size of well above 3 mm [12]. XeCl excimer-laser light with wavelength of 308 nm and pulse duration of 66 ns (XMR 7100 system) was irradiated onto the a-Si ®lm on the glass substrate. A pulsed excimerlaser was focused on the substrate and irradiated with 100 pulses. The laser scanned across the substrate with step and repeat manner using an optical scanning system mounted on a X±Y stage (Fig. 3).
The ®lter characteristics of the two (poly)crystalline structures (Fig. 5) and of the amorphous material were measured. The a-Si structure indeed behaves as a low pass ®lter (Fig. 6), but has a moderate steepness. Although it suppresses UV light
4. Experimental setup A thin layer of Si forms a short-wavelength ®lter in the sense that visible radiation passes through the layer without substantial attenuation, while light in the UV range is highly absorbed. This is particularly useful for measurements of molecules that can be excited in the UV and emit (¯uoresce) in the visible part of the spectrum. A ®lter placed on the top of a photodiode forms a protective shield preventing the
Fig. 4. The measurement setup for filter evaluation.
Fig. 5. a-Si film (75 nm) on a Pyrex wafer. The 1 cm2 1 cm2 squares are re-crystallized areas exhibiting higher transparency for visible light.
Fig. 6. Comparison between a-Si (measured and simulated results) and (poly)crystalline Si (simulated).
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Fig. 7. Comparison between measured and simulated results for thin-film (poly)crystalline silicon layers.
strongly, it exhibits considerable suppression of light in the visible spectrum as well. Fig. 7 displays the transmittance of the (poly)crystalline ®lters compared to the simulation results. The crystalline ®lter acts as a low pass ®lter with much steeper roll-off and a well-de®ned cut-off frequency at 400 nm. This effect is caused by the reduced lattice disorder in a crystalline material, which causes the reduction in extinction coef®cient k at energy levels just below the direct bandgap to be more abrupt in comparison to amorphous material. As photon energy reaches the direct bandgap threshold (3.4 eV, 380 nm) direct electron excitation becomes possible, and the absorption increases greatly. As shown in Figs. 6 and 7, the thin-®lm crystalline silicon ®lter exhibits large suppression of the UV light. At the same time, suppression of light in the visible spectrum is slight and allows weak visible light to be distinguished in the presence of a strong background (UV) signal. 6. NADH detection A variety of enzyme reactions can be monitored using the conversion of NAD to its ¯uorescent product NADH.
Fig. 8. Measurement setup for selective NADH fluorescence analysis.
We are directing our efforts towards designing a sensor array for NADH concentration measurements in each reactor of the array. NADH absorbs light of wavelength 340 nm and radiates secondary (¯uorescence) photons at a wavelength of 450 nm. The NADH measurements were conducted with the setup shown in Fig. 8. The cuvette containing the NADH, the optical ®lter and the detector (a calibrated Hamamatsu S1226 diode) were stacked together as shown in the ®gure. The NADH solution was exposed to light from the argon laser. Because of the relatively high power of the laser, much of the incident UV-light (10 mW) passed straight through the cuvette (the optical path in the cuvette is only 1 mm) and reached the detector. Using the optical thin-®lm polycrystalline silicon ®lter, NADH ¯uorescence measurements were performed. Fig. 9 shows the photodiode detector current to varying concentrations of the NADH solution. As shown in the ®gure, up to 1 mMol/l the response of the photodetector is linearly proportional to the concentration. It can be seen that the response without ®lter is almost constant with varying concentration. Slight sloping is observed due to absorption phenomena.
Fig. 9. Measured photodiode detector current for different concentrations of NADH.
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7. Conclusions We have developed a small-wavelength rejection optical ®lter for NADH ¯uorescence analysis in subnanoliter arrays for HSS. The ®lter consists of a thin re-crystallized silicon layer. The optical properties of the ®lter are such that the ¯uorescent light of wavelengths of 400 nm or higher can be measured in the presence of the (UV) excitation light (in the case of NADH molecules 340±360 nm) with 35 dB selectivity. By increasing the thickness of the silicon ®lms the selectivity can be increased to more than 50 dB. This will enable the detection of low concentrations of an analyte (¯uorescence measurement) without applying sophisticated and expensive optical equipment. Measurements of NADH solvents with concentrations in the range 0±1 mMol/l, based on a system containing a crystalline Si ®lter on a glass substrate have been obtained. The ®lters investigated are compatible with available CMOS processes and are, therefore, very easy to implement on a chip in combination with integrated electronics by using post-processing. Silicon thin®lm ®lters are suitable for detection of various ¯uorophores used in biochemical analysis, which are excited by UV light and ¯uoresce in the visible spectrum (e.g. NADH, NADPH, Dansyl Cl, Flavins). Other thin-®lm materials having different optical properties and layer thickness could be useful for other ¯uorophores. Acknowledgements The authors would like to thank A.J. van der Lingen (Optic Research Group, Department of Applied Physics, TUDelft) for his kind cooperation concerning the UV measurements, R. Moerman (Kluyvert Laboratory for Biotechnology, TUDelft) for supplying the NADH solutions, J. Bastemeijer, J. Nieuwenhuis and B. Gray (TUDelft, Electronic Instrumentation Laboratory) for the inspiring discussions and A. Berthold (DIMES Technology Center) for producing the structures researched. This research is supported by the Delft Interfaculty Research Center (DIOC), Intelligent Molecular Diagnostic Systems (IMDS). References [1] S.K. Moore, Making chips, IEEE Spectrum 38 (March 2001) 54±60. [2] K.T. Hjelt, P. Szczaurski, L.R.v.d. Doel, W. Lubking, B. Jakoby, M.J. Vellekoop, Measurement of liquid volumes in sub-nanoliter reactors, in: Proceedings of the Transducers'99, Sendai, Japan, June 1999, pp. 748±751. [3] R.A. Clark, P.B. Hietpas, A.G. Ewing, Electrochemical analysis in picoliter microvials, Anal. Chem. 69 (1997) 256±263. [4] D. Taylor, A. Waggoner, R. Murphy, F. Lanni, R. Birge, Application of Fluorescence in the Biomedical Sciences, Liss, New York, 1986. [5] L.R.v.d. Doel, M.J. Vellekoop, P.M. Sarro, S. Picioreanu, R. Moerman, H. Frank, G.K. van Dedem, K. Hjelt, I.T. Young, Fluorescence detection in subnanoliter microarrays, Proc. SPIE 3606 (1999) 28±39.
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[6] D.P. Poenar, Thin film color sensors, Ph.D. Thesis, Delft University Press, Delft, 1996. [7] D.P. Poenar, R.F. Wolffenbuttel, Optical properties of thin-film siliconcompatible materials, Appl. Optics 36 (21) (1997) 5122±5128. [8] A. Pauchard, P.A. Besse, M. Bartek, R.F. Wolffenbuttel, R.S. Popovic, Ultraviolet-selective avalanche photodiode, Sensors and Actuators A 82 (1±3) (2000) 128±134. [9] J. Singh, Semiconductor OptoelectronicsÐPhysics and Technology, McGraw-Hill International Edition, Electrical Engineering Series, McGraw-Hill, New York, 1995, pp. 170±202. [10] T. Sameshima, S. Usui, M. Sekiya, XeCl excimer laser annealing used in fabrication of poly-Si TFTs, IEEE Electron Dev. Lett. 7 (1986) 276. [11] J.S. Im, H.J. Kim, M.O. Thompson, Phase transformation mechanisms involved on excimer laser, Appl. Phys. Lett. 63 (1993) 196. [12] H. Kuriyama, T. Nohda, S. Ishida, T. Kuwahara, S. Noguchi, S. Kitayama, S. Tsuda, S. Ankano, Lateral grain growth of poly-Si films with a specific orientation by an excimer laser annealing method, Jpn. J. Appl. Phys. 32 (1993) 6190.
Biographies V.P. Iordanov was born in 1975 in Kustendil, Bulgaria. He obtained his degree in Applied Physics from Sofia University, Bulgaria, in 1999. The same year, he came to the Delft University of Technology under the Tempus Student Exchange Program to prepare his diploma in the field of electronics design. He received his MSc degrees in the fields of Microelectronics and Information Technologies and Electronics Design, from the Sofia University in 1999. Since November 1999, the Faculty of Applied Sciences, Department of Applied Physics has employed him as a PhD student. His project involves development of optical sensors and devices. His main research interests are in the field of physical sensors, microelectronics and interface design. G.W. Lubking was born in 1944 in Rotterdam, the Netherlands. He received the MSc degree in 1967 from the Delft University of Technology and worked there since 1969. He was a senior electronics designer responsible for the design of electronic circuitry for various sensors. Tragically, G.W. Lubking has passed away suddenly on the 18 December 2000. R. Ishihara was born in 1967 in Japan. He received the BE, ME and PhD degrees from Department of Physical Electronics, Tokyo Institute of Technology, Japan, in 1991, 1993 and 1996, respectively. From 1990 to 1996, he was a research member with Professor M. Matsumura in Department of Physical Electronics, Tokyo Institute of Technology. His research activities were low-temperature deposition of silicon nitride film and excimer-laser crystallization of thin silicon films for thin-film transistor application. Since April 1996, he has been with Delft Institute of Microelectronics and Submicron Technology (DIMES), at the Delft University of Technology, where he is a member of the Laboratory Electronic Components, Technology and Materials as a senior research scientist. He is in charge of a number of projects related to laser crystallization and thin-film transistor technologies. R.F. Wolffenbuttel received his MSc degree in 1984 and PhD degree in 1988, both from the Delft University of Technology. Between 1986 and 1993 he has been an Assistant Professor and since 1993, he has been an Associate Professor at the Laboratory of Electronic Instrumentation of the Delft University of Technology and is involved in instrumentation and measurement in general and on-chip functional integration of microelectronic circuits and silicon sensor, fabrication compatibility issues and micromachining in silicon and microsystems in particular. He was a visitor at the University of Michigan, Ann Arbor, USA, in 1992, 1999 and 2001, Tohoku University, Sendai, Japan in 1995 and EPFL Lausanne,
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Switzerland, in 1997. He is the recipient of a 1997 NWO Pionier award. He served as general chairman of the Dutch National Sensor Conference in 1996 and Eurosensors in 1999. P.M. Sarro received the Laurea degree in solid-state physics from the University of Naples, Italy, in 1980. From 1981 to 1983, she was a postdoctoral fellow in the Photovoltaic Research Group of the Division of Engineering, Brown University, Rhode Island, USA, where she worked on thin-film photovoltaic cell fabrication by chemical spray pyrolysis. In 1987, she received the PhD degree in Electrical Engineering at the Delft University of Technology, the Netherlands, her thesis dealing with infrared sensors based on integrated silicon thermopiles. Since then, she has been with the Delft Institute of Microelectronics and Submicron Technology (DIMES), at the Delft University, where she is responsible for research on integrated silicon sensors and microsystems technology. Since April 1996, she is also an Associate Professor in the Electronic Components, Materials and Technology Laboratory of the Delft University. She is an IEEE member since 1984 and a Senior Member since 1997. She acts as reviewer
for several technical journals and she has served as technical program committee member of the ESSDERC conferences (since 1995), the SPIE Fifth Annual Symposium on SMART STRUCTURES and MATERIAL'98 and EUROSENSORS'99 and '00. M.J. Vellekoop was born in Amsterdam in 1960. He received the BSc degree in Physics in 1982 and the PhD degree in Electrical Engineering in 1994. In 1984 he joined the Delft University of Technology to work in the field of acoustic wave sensors. In 1988 he co-founded Xensor Integration BV, a company specialized in the development and production of integrated sensors, where he was Managing Director from 1988 to 1996. From 1996 till the summer of 2001 he was leading the Integrated Physical Chemosensors group of the DIMES Electronic Instrumentation Laboratory (Microelectronics Department, Delft University of Technology), since 1997 as an Associate Professor. Since the summer of 2001 he is a Full Professor at the Institute of Industrial Electronics and Material Science at the Vienna University of Technology, Austria, in the field of industrial sensor systems.
Silicon thin-®lm UV ®lter for NADH ¯uorescence analysis V.P. Iordanova,*, G.W. Lubkinga, R. Ishiharab, R.F. Wolffenbuttela, P.M. Sarrob, M.J. Vellekoopc a
Electronic Instrumentation Laboratory, Delft University of Technology, DIMES, Mekelweg 4, 2628 Delft, The Netherlands Electronic Components, Technology and Materials Laboratory, Delft University of Technology, DIMES, Delft, The Netherlands c Institute of Industrial Electronics and Material Science, Vienna University of Technology, Vienna, Austria
b
Received 1 June 2001; received in revised form 2 November 2001; accepted 14 November 2001
Abstract A new optical detector system for ¯uorescence analysis in on-chip high-speed screening arrays is described. It consists of a photodiode covered by a re-crystallized silicon ®lm, which acts as a single-®lm optical ®lter. The crystalline structure of the silicon ®lm has a strong in¯uence on the optical properties of the ®lter. The fabrication process and characterization of a polycrystalline Si thin-®lm on glass are reported. The method is compatible with standard CMOS processes. The optical properties of the designed ®lter are such that ¯uorescent light of wavelengths of 420 nm or higher can be measured in the presence of (UV) excitation light (340±360 nm). A selectivity exceeding 35 dB has been demonstrated. The technique has the potential for 50 dB selectivity and higher. NADH concentration measurements in the range 0±1 mMol/l illustrate the application of the system for determination of enzymatic activity of different analytes. # 2002 Elsevier Science B.V. All rights reserved. Keywords: UV ®lter; Fluorescence; Biosensor; High-speed screening
1. Introduction High-speed screening (HSS) on arrays implemented in silicon substrates is one of the new tools investigated for biochemical analysis. Such devices consist of an array of subnanoliter wells in which chemical or biochemical samples are examined. By carrying out assays in small volumes [1±3], signi®cant savings can be achieved in the cost of targets, compounds and reagents. The miniaturization of an assay screen also allows more measurements to be carried out in parallel in the same time. Such subnanoliter reactor arrays use one million times less sample than the commonly used 96-reactor titer plates. Fluorescence is an attractive method of detection in assay analyses. It has been used for monitoring of different enzymatic reactions and for a variety of compounds including, but not limited to, pesticides, vitamins and amino acids [4]. Fluorescence detection is a ``low background'' technique, as opposed to absorbance. In an absorbance detector, the measured signal is related to the difference in light intensity with a sample present versus the signal in the absence of a sample. For lower levels of analyte, this difference becomes
* Corresponding author. Tel.: 31-15-278-3342; fax: 31-15-278-5755. E-mail address: [email protected] (V.P. Iordanov).
increasingly smaller, and therefore increasingly harder to detect. The ¯uorescence detector, however, measures light emitted from the solvent in an otherwise dark background. The result is a much lower detection limit, limited by the electronic noise and the dark current of the detector. The other primary advantage of ¯uorescence is selectivity. The two major reasons for greater selectivity, as opposed to absorbance detection are: (1) most organic molecules absorb ultraviolet (UV)/visible light but, not all ¯uoresce; (2) the wavelength of excitation light is in a different spectral range, compared to the emission spectrum. Many ¯uorescence detectors are commercially available today. Each has its own instrumental variables, and is optimized for a certain application. In our silicon subnanoliter array, the wells can be read out in parallel by using a lens and camera [5], but this method is bulky and expensive. An attractive solution is to equip each well with its own photodetector. Using monolithic integration for fabricating the array makes it possible to do the screening electronically. This makes higher speed scanning possible in comparison with mechanical screening. A variety of enzyme reactions can be monitored using the conversion of NAD (nicotinamide adenine dinucleotide) to its ¯uorescent product NADH. We are directing our efforts towards designing a sensor array for NADH concentration measurements in each well. NADH absorbs light of wavelength 340 nm and radiates secondary (¯uorescence)
0924-4247/02/$ ± see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 4 - 4 2 4 7 ( 0 1 ) 0 0 8 4 8 - 2
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Fig. 1. Schematic cross-view of a subnanoliter reactor including a photodetector.
photons with a wavelength of 450 nm. For ¯uorescence detection each reactor cell contains a photo-sensor at the bottom. A possible method to illuminate the analyte is by irradiating the reactor chambers from the side by means of optical waveguides. Such a con®guration is attractive for a single chamber reactor, but in the case of an array a complicated network of waveguides is required in order to ensure that every well receives an equal amount of primary light. A con®guration where the sensor array is illuminated from above (Fig. 1) is the simplest from a technological point of view and allows the use of very simple optical equipment. However, a large fraction of the primary light will pass through the analyte and reach the detector. This light will cause a strong background signal. Therefore, an optical ®lter is required in order to suppress the undesired primary light. Although it is possible to place the junction of the photodiode such that it is sensitive to a selected spectral range only, this would not be suf®cient to provide the required selectivity [6]. As discussed in the text below, silicon appears to exhibit the properties needed to form a UV ®lter [7,8]. Furthermore, a Si-based ®lter structure can be implemented using existing chip processing technology. 2. Optical absorption in Si The absorption process for photons with energy near the bandgap is strongest in direct bandgap semiconductors since the photon can directly cause a transition of an electron from the valence band to the conduction band. When a semiconductor does not have a direct bandgap, absorption of a photon only takes place if a phonon (or lattice vibration) participates in the process. The absorption coef®cient of photons in crystalline Si, an indirect semiconductor, is lower than that of a direct material
Fig. 2. Absorption coefficient of silicon.
and rises only slowly at energy levels near the bandgap (1.12 eV). Once the photon energy is suf®cient to cross the direct gap region (3.4 eV), the absorption coef®cient increases rapidly since direct transitions are possible. Apart from phonon scattering there are also other scattering mechanisms such as alloy scattering and impurity scattering, which can cause optical absorption in indirect semiconductors. As a result, ``poor'' quality indirect semiconductors have a higher absorption coef®cient than pure indirect materials (Fig. 2). In amorphous Si (a-Si), e.g. the absorption coef®cient is quite strong and its absorption properties resemble those of a material with a direct bandgap of 1.12 eV [9]. 3. Laser annealing of a-Si layer Poly-Si ®lms were formed on glass by irradiation of excimer-laser light on an a-Si ®lm with thickness of 75 mm. Excimer-laser basically uses emission of deep UV light from excited ``dimmer'' of rare gas halide, such as XeCl, XeF and ArF. The excimer-laser light has unique characteristics such as a short-wavelength ranged from 150 to 350 nm, a short pulse width of a few tenths of nanosecond, and high output power of about 1 J. Since Si has a strong absorption of the deep UV light, the absorption is limited to a very thin layer of a few tenths of nanometer, where heat is generated by lattice vibration. Since the pulse duration is very short, the penetration of the heat in the substrate is very shallow. The excimer-laser has been used for melting of thin a-Si layer on substrate, such as glass, and to transform it into (poly)crystalline Si [10]. The typically obtained grain size of the excimer-laser crystallized poly-Si is about 100 nm. The grain size takes the maximum value of about 1 mm with the laser energy which Si ®lm is almost completely melted with [11]. By applying
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penetration of the undesired primary (UV) light, while permitting the passage of the secondary (¯uorescence) light. The optical properties of thin-®lm layers (transmittance and absorption) were simulated with TFCalcÐThin-Film Design Software, Version 2.9, Software Spectra. In order to verify the results, the optical transmittance of amorphous and crystalline silicon was measured. The measurements are made with the setup shown in Fig. 4 using a calibrated Hamamatsu S1226 diode. An additional measurement in the UV range was performed using an argon laser, Spectra Physics, Model 2020-05, l 365 nm, optical power 240 mW cm 2. 5. Filter experiments
Fig. 3. XeCl excimer-laser setup for a-Si re-crystallization.
multi-shot irradiation, the substantial grain growth is induced, which yields to grain size of well above 3 mm [12]. XeCl excimer-laser light with wavelength of 308 nm and pulse duration of 66 ns (XMR 7100 system) was irradiated onto the a-Si ®lm on the glass substrate. A pulsed excimerlaser was focused on the substrate and irradiated with 100 pulses. The laser scanned across the substrate with step and repeat manner using an optical scanning system mounted on a X±Y stage (Fig. 3).
The ®lter characteristics of the two (poly)crystalline structures (Fig. 5) and of the amorphous material were measured. The a-Si structure indeed behaves as a low pass ®lter (Fig. 6), but has a moderate steepness. Although it suppresses UV light
4. Experimental setup A thin layer of Si forms a short-wavelength ®lter in the sense that visible radiation passes through the layer without substantial attenuation, while light in the UV range is highly absorbed. This is particularly useful for measurements of molecules that can be excited in the UV and emit (¯uoresce) in the visible part of the spectrum. A ®lter placed on the top of a photodiode forms a protective shield preventing the
Fig. 4. The measurement setup for filter evaluation.
Fig. 5. a-Si film (75 nm) on a Pyrex wafer. The 1 cm2 1 cm2 squares are re-crystallized areas exhibiting higher transparency for visible light.
Fig. 6. Comparison between a-Si (measured and simulated results) and (poly)crystalline Si (simulated).
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Fig. 7. Comparison between measured and simulated results for thin-film (poly)crystalline silicon layers.
strongly, it exhibits considerable suppression of light in the visible spectrum as well. Fig. 7 displays the transmittance of the (poly)crystalline ®lters compared to the simulation results. The crystalline ®lter acts as a low pass ®lter with much steeper roll-off and a well-de®ned cut-off frequency at 400 nm. This effect is caused by the reduced lattice disorder in a crystalline material, which causes the reduction in extinction coef®cient k at energy levels just below the direct bandgap to be more abrupt in comparison to amorphous material. As photon energy reaches the direct bandgap threshold (3.4 eV, 380 nm) direct electron excitation becomes possible, and the absorption increases greatly. As shown in Figs. 6 and 7, the thin-®lm crystalline silicon ®lter exhibits large suppression of the UV light. At the same time, suppression of light in the visible spectrum is slight and allows weak visible light to be distinguished in the presence of a strong background (UV) signal. 6. NADH detection A variety of enzyme reactions can be monitored using the conversion of NAD to its ¯uorescent product NADH.
Fig. 8. Measurement setup for selective NADH fluorescence analysis.
We are directing our efforts towards designing a sensor array for NADH concentration measurements in each reactor of the array. NADH absorbs light of wavelength 340 nm and radiates secondary (¯uorescence) photons at a wavelength of 450 nm. The NADH measurements were conducted with the setup shown in Fig. 8. The cuvette containing the NADH, the optical ®lter and the detector (a calibrated Hamamatsu S1226 diode) were stacked together as shown in the ®gure. The NADH solution was exposed to light from the argon laser. Because of the relatively high power of the laser, much of the incident UV-light (10 mW) passed straight through the cuvette (the optical path in the cuvette is only 1 mm) and reached the detector. Using the optical thin-®lm polycrystalline silicon ®lter, NADH ¯uorescence measurements were performed. Fig. 9 shows the photodiode detector current to varying concentrations of the NADH solution. As shown in the ®gure, up to 1 mMol/l the response of the photodetector is linearly proportional to the concentration. It can be seen that the response without ®lter is almost constant with varying concentration. Slight sloping is observed due to absorption phenomena.
Fig. 9. Measured photodiode detector current for different concentrations of NADH.
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7. Conclusions We have developed a small-wavelength rejection optical ®lter for NADH ¯uorescence analysis in subnanoliter arrays for HSS. The ®lter consists of a thin re-crystallized silicon layer. The optical properties of the ®lter are such that the ¯uorescent light of wavelengths of 400 nm or higher can be measured in the presence of the (UV) excitation light (in the case of NADH molecules 340±360 nm) with 35 dB selectivity. By increasing the thickness of the silicon ®lms the selectivity can be increased to more than 50 dB. This will enable the detection of low concentrations of an analyte (¯uorescence measurement) without applying sophisticated and expensive optical equipment. Measurements of NADH solvents with concentrations in the range 0±1 mMol/l, based on a system containing a crystalline Si ®lter on a glass substrate have been obtained. The ®lters investigated are compatible with available CMOS processes and are, therefore, very easy to implement on a chip in combination with integrated electronics by using post-processing. Silicon thin®lm ®lters are suitable for detection of various ¯uorophores used in biochemical analysis, which are excited by UV light and ¯uoresce in the visible spectrum (e.g. NADH, NADPH, Dansyl Cl, Flavins). Other thin-®lm materials having different optical properties and layer thickness could be useful for other ¯uorophores. Acknowledgements The authors would like to thank A.J. van der Lingen (Optic Research Group, Department of Applied Physics, TUDelft) for his kind cooperation concerning the UV measurements, R. Moerman (Kluyvert Laboratory for Biotechnology, TUDelft) for supplying the NADH solutions, J. Bastemeijer, J. Nieuwenhuis and B. Gray (TUDelft, Electronic Instrumentation Laboratory) for the inspiring discussions and A. Berthold (DIMES Technology Center) for producing the structures researched. This research is supported by the Delft Interfaculty Research Center (DIOC), Intelligent Molecular Diagnostic Systems (IMDS). References [1] S.K. Moore, Making chips, IEEE Spectrum 38 (March 2001) 54±60. [2] K.T. Hjelt, P. Szczaurski, L.R.v.d. Doel, W. Lubking, B. Jakoby, M.J. Vellekoop, Measurement of liquid volumes in sub-nanoliter reactors, in: Proceedings of the Transducers'99, Sendai, Japan, June 1999, pp. 748±751. [3] R.A. Clark, P.B. Hietpas, A.G. Ewing, Electrochemical analysis in picoliter microvials, Anal. Chem. 69 (1997) 256±263. [4] D. Taylor, A. Waggoner, R. Murphy, F. Lanni, R. Birge, Application of Fluorescence in the Biomedical Sciences, Liss, New York, 1986. [5] L.R.v.d. Doel, M.J. Vellekoop, P.M. Sarro, S. Picioreanu, R. Moerman, H. Frank, G.K. van Dedem, K. Hjelt, I.T. Young, Fluorescence detection in subnanoliter microarrays, Proc. SPIE 3606 (1999) 28±39.
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[6] D.P. Poenar, Thin film color sensors, Ph.D. Thesis, Delft University Press, Delft, 1996. [7] D.P. Poenar, R.F. Wolffenbuttel, Optical properties of thin-film siliconcompatible materials, Appl. Optics 36 (21) (1997) 5122±5128. [8] A. Pauchard, P.A. Besse, M. Bartek, R.F. Wolffenbuttel, R.S. Popovic, Ultraviolet-selective avalanche photodiode, Sensors and Actuators A 82 (1±3) (2000) 128±134. [9] J. Singh, Semiconductor OptoelectronicsÐPhysics and Technology, McGraw-Hill International Edition, Electrical Engineering Series, McGraw-Hill, New York, 1995, pp. 170±202. [10] T. Sameshima, S. Usui, M. Sekiya, XeCl excimer laser annealing used in fabrication of poly-Si TFTs, IEEE Electron Dev. Lett. 7 (1986) 276. [11] J.S. Im, H.J. Kim, M.O. Thompson, Phase transformation mechanisms involved on excimer laser, Appl. Phys. Lett. 63 (1993) 196. [12] H. Kuriyama, T. Nohda, S. Ishida, T. Kuwahara, S. Noguchi, S. Kitayama, S. Tsuda, S. Ankano, Lateral grain growth of poly-Si films with a specific orientation by an excimer laser annealing method, Jpn. J. Appl. Phys. 32 (1993) 6190.
Biographies V.P. Iordanov was born in 1975 in Kustendil, Bulgaria. He obtained his degree in Applied Physics from Sofia University, Bulgaria, in 1999. The same year, he came to the Delft University of Technology under the Tempus Student Exchange Program to prepare his diploma in the field of electronics design. He received his MSc degrees in the fields of Microelectronics and Information Technologies and Electronics Design, from the Sofia University in 1999. Since November 1999, the Faculty of Applied Sciences, Department of Applied Physics has employed him as a PhD student. His project involves development of optical sensors and devices. His main research interests are in the field of physical sensors, microelectronics and interface design. G.W. Lubking was born in 1944 in Rotterdam, the Netherlands. He received the MSc degree in 1967 from the Delft University of Technology and worked there since 1969. He was a senior electronics designer responsible for the design of electronic circuitry for various sensors. Tragically, G.W. Lubking has passed away suddenly on the 18 December 2000. R. Ishihara was born in 1967 in Japan. He received the BE, ME and PhD degrees from Department of Physical Electronics, Tokyo Institute of Technology, Japan, in 1991, 1993 and 1996, respectively. From 1990 to 1996, he was a research member with Professor M. Matsumura in Department of Physical Electronics, Tokyo Institute of Technology. His research activities were low-temperature deposition of silicon nitride film and excimer-laser crystallization of thin silicon films for thin-film transistor application. Since April 1996, he has been with Delft Institute of Microelectronics and Submicron Technology (DIMES), at the Delft University of Technology, where he is a member of the Laboratory Electronic Components, Technology and Materials as a senior research scientist. He is in charge of a number of projects related to laser crystallization and thin-film transistor technologies. R.F. Wolffenbuttel received his MSc degree in 1984 and PhD degree in 1988, both from the Delft University of Technology. Between 1986 and 1993 he has been an Assistant Professor and since 1993, he has been an Associate Professor at the Laboratory of Electronic Instrumentation of the Delft University of Technology and is involved in instrumentation and measurement in general and on-chip functional integration of microelectronic circuits and silicon sensor, fabrication compatibility issues and micromachining in silicon and microsystems in particular. He was a visitor at the University of Michigan, Ann Arbor, USA, in 1992, 1999 and 2001, Tohoku University, Sendai, Japan in 1995 and EPFL Lausanne,
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Switzerland, in 1997. He is the recipient of a 1997 NWO Pionier award. He served as general chairman of the Dutch National Sensor Conference in 1996 and Eurosensors in 1999. P.M. Sarro received the Laurea degree in solid-state physics from the University of Naples, Italy, in 1980. From 1981 to 1983, she was a postdoctoral fellow in the Photovoltaic Research Group of the Division of Engineering, Brown University, Rhode Island, USA, where she worked on thin-film photovoltaic cell fabrication by chemical spray pyrolysis. In 1987, she received the PhD degree in Electrical Engineering at the Delft University of Technology, the Netherlands, her thesis dealing with infrared sensors based on integrated silicon thermopiles. Since then, she has been with the Delft Institute of Microelectronics and Submicron Technology (DIMES), at the Delft University, where she is responsible for research on integrated silicon sensors and microsystems technology. Since April 1996, she is also an Associate Professor in the Electronic Components, Materials and Technology Laboratory of the Delft University. She is an IEEE member since 1984 and a Senior Member since 1997. She acts as reviewer
for several technical journals and she has served as technical program committee member of the ESSDERC conferences (since 1995), the SPIE Fifth Annual Symposium on SMART STRUCTURES and MATERIAL'98 and EUROSENSORS'99 and '00. M.J. Vellekoop was born in Amsterdam in 1960. He received the BSc degree in Physics in 1982 and the PhD degree in Electrical Engineering in 1994. In 1984 he joined the Delft University of Technology to work in the field of acoustic wave sensors. In 1988 he co-founded Xensor Integration BV, a company specialized in the development and production of integrated sensors, where he was Managing Director from 1988 to 1996. From 1996 till the summer of 2001 he was leading the Integrated Physical Chemosensors group of the DIMES Electronic Instrumentation Laboratory (Microelectronics Department, Delft University of Technology), since 1997 as an Associate Professor. Since the summer of 2001 he is a Full Professor at the Institute of Industrial Electronics and Material Science at the Vienna University of Technology, Austria, in the field of industrial sensor systems.