- Email: [email protected]
Diamond micro system for bio-chemistry
Diamond and Related Materials 10 Ž2001. 722᎐730
Diamond micro system for bio-chemistry M. Adamschik a,U , M. Hinz c , C. Maier b, P. Schmida , H. Seliger c , E.P. Hofer b, E. Kohna a
b
Department of Electron De¨ ices and Circuits, Uni¨ ersity of Ulm, D-89081 Ulm, Germany Department of Measurement Control and Microtechnology, Uni¨ ersity of Ulm, Control and Microtechnology, D-89081 Ulm, Germany c Department of Polymers, Uni¨ ersity of Ulm, D-89081 Ulm, Germany
Abstract This work illustrates the potential of diamond micro system technologies progressively developed in the last years for an application in the bio-chemical field. A diamond micro reactor system based on a novel integration concept is presented and the role of diamond in this generic system is described. It consists of reaction chambers with removable bottom and integrated micro dosage elements allowing the ejection and mixture of two different fluids onto the removable bottom substrate. As an example, this system is used in a novel DNA-synthesis cycle. In this application the diamond micro reactor system combined with a specifically designed chemistry for the DNA-synthesis enables the parallel production of DNA-chain-clusters with individual sequence arranged in an array ŽDNA-Chip.. 䊚 2001 Elsevier Science B.V. All rights reserved. Keywords: Diamond micro system; Bio-chemistry; DNA; Diamond MEMS; Diamond actuators
1. Introduction Many diamond-based sensor and actuator structures like piezoresistive- w1,2x, pressure- w3,4x, accelerationw5x, temperature- w6,7x, UV-sensors w8x, micro switches w9,10x and various micro electromechanical system ŽMEMS. applications w11᎐13x have been reported previously. Beyond these applications and the application as electrochemical electrode w14᎐17x in the present work the potential of diamond for devices in the new field of bio-chemistry and nano-chemistry shall be demonstrated. The role of diamond in a generic chemical micro reactor system w19x will be described. Chemical micro reactors have emerged as an essential part of all bio-chemical synthesis and analysis systems w20᎐22x.
U
Corresponding author. University of Ulm, Albert-Einstein-Allee 45, D-89081 Ulm, Germany. Tel.: q49-731-502-6179; fax: q49-731502-6155. E-mail address: [email protected] ŽM. Adamschik..
The system presented here consists of three main parts ŽFig. 1.: 1. an overlay carrier for electrical and liquid supply; 2. a micro dosage unit based on the bubble jet principle combined with an integrated array of reaction chambers with open bottom; and 3. a removable substrate representing the bottom of the reaction chamber array. Integrated into the micro dosage unit are individually controllable diamond bubble-jet elements for precise injection of two different fluids into the reaction chambers. The diamond bubble-jet element has in essence already been published previously w5,18,23᎐25x and is shown in Fig. 2. This element combines high robustness, good chemical inertness and simple technology, thus serving as a reliable work horse for integration into such a system. The bubble-jet element had already been character-
0925-9635r01r$ - see front matter 䊚 2001 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 5 - 9 6 3 5 Ž 0 0 . 0 0 6 1 4 - 2
M. Adamschik et al. r Diamond and Related Materials 10 (2001) 722᎐730
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Fig. 1. Schematic cross-section of micro reactor system.
ized extensively, showing its outstanding properties w27,28x. Due to the high hardness, high mechanical strength and chemical inertness no passivation layers are necessary on top of the diamond heater element to prevent cavitation damages or chemical attack of the heater surface resulting in high reliability, low thermal induced stresses, small heat losses, and a simple technology. The low specific heat capacity allows fast temperature changes from room temperature up to the spinodalean limit Ž312⬚C for water w24x. necessary for proper overheating and bubble nucleation of the fluids. The good electrical isolating properties of undoped diamond combined with the high thermal conductivity allows electrically insulating layers w29x on top of the heater surface to prevent leakage currents between individual heaters during the ejection of acidic or basic solutions. From lifetime tests over 10 8 nucleation cycles could be extracted without deterioration showing that the heater element itself is not limiting the lifetime w25x. Due to the material properties the diamond heaters are able to withstand power densities up to 30 GWrm2 w25x being 10 times that of the nucleation power density in a normal operation. Fig. 3 shows an example of the electrical power applied onto a 60 = 60m heater element vs. minimum heating time for nucleation using a water covered heater element. Here the maximum power of 30 GWrm2 has been applied, resulting in an ultra-fast sub-microsecond nucleation time. Fig. 4 shows the time for damage for this extremely high power level, which is approximately four times of that necessary for nucleation. The new integrated diamond micro reactor system discussed in the following contains two bubble-jet elements in a single reaction chamber with a heater area of 30 = 30 m to overheat the liquids ŽFig. 5.. The reaction chambers are arranged as an array. The micro dosage unit is bonded into an overlay carrier, which contains the liquid and electrical connections. The open bottom of the reaction chamber array can be pressed
onto the surface of a planar or patterned reaction substrate, which can be removed for inspection after the chemical reaction took place. The amount of ejected liquid can be adjusted very precisely by sequentially driving the heater elements, resulting in a digital increase by 30-pl increments per drop w24x. Thus, the system enables tight control of chemical exotherm or endotherm reactions, mixing, etc. Due to the chemical inertness, the parallel arrangement of the reaction chambers, the selectively addressable heater elements and the precisely adjustable volumes of ejected liquids, this diamond chip represents a generic building block for nano-chemical systems. As a first example the synthesis of oligonucleotides Žoligonucleotide s part of a DNA-chain, see Fig. 9b. will be presented and discussed. The system allows fast and parallel fabrication of oligonucleotides with individual sequences Ž‘custom design’. on the surface of a reaction substrate by combining a specially designed chemistry w26x. Due to the aggressive nature of the chemistry all major parts of the system need to be chemically inert.
Fig. 2. Diamond bubble-jet ejector element.
Fig. 3. Applied electrical heating power vs. nucleation time w25x.
2. Technology The heart of the system is the micro dosage unit ŽFig. 1, part II. consisting of an array of open bottom reaction chambers with integrated bubble-jet injectors. It is fabricated from two parts as illustrated in Fig. 5a,b
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M. Adamschik et al. r Diamond and Related Materials 10 (2001) 722᎐730
Fig. 4. Applied electrical heating power vs. time until destruction w25x.
using two different diamond-on-Si wafers. The assembled part Žpart 1 and 2 in Fig. 5. is then mounted into the fluidic and electrical supply carrier. Each micro reaction chamber contains two ‘bubblejet’ injector elements as indicated above, based on highly doped diamond micro heater elements ŽFig. 5a.. The diamond heater elements are grown by microwave plasma CVD onto a thermal insulation layer and are doped by boron. After the growth step dry etching was used for patterning. For the supply and the distribution of the reaction fluids on this part a photosensitive and chemical resistant polyimide capillary wall system was used, because this part of the device is not thermally stressed. The second part consists of the diamond nozzle plate and reaction chambers ŽFig. 5b.. The rectangular reaction chamber geometry was formed by anisotropic silicon etching using KOH. In the etching process diamond again offers the advantage of a selective etch stop. A 5-m thick CVD-diamond film was grown by PECVD and holes with a diameter of 30 m were etched by dry etching. In the last step the nozzle plate ᎏ reaction chamber part is bonded onto the top of the capillary wall system of part 1 forming a leak-tight microfluidic system. To connect the micro fluidic capillary system with conventional tubes an overlay carrier was fabricated into which the micro reaction chip was
bonded. It contains an integrated fluidic system, fluidic connectors and electrical connectors. Fig. 5c represents a schematic view of a single reaction chamber of the complete array. Fig. 6a shows part 1 of the device and a detailed view of one heater element. Fig. 7 shows part 2, the nozzle plate with integrated reaction chambers and a detailed SEM micrograph of a single nozzle. Anisotropic and smooth sidewalls can be seen, preventing vortexes during drop ejection. Fig. 8a shows a device with 10 reaction chambers in a one-dimensional array after chip bonding of part 1 and 2. Fig. 8b depicts a top view into a single reaction chamber showing the nozzles, the metallization and the diamond heater elements through the transparent CVD-diamond nozzle plate. The completely assembled system with the high-grade steel carrier and its electrical connections can be seen in Fig. 8c.
3. Application As the first application the synthesis of oligonucleotides, which are a segment of a DNA-chain, is discussed. Such a synthesis is carried out in a cycle of four steps. The synthesis is supposed to be controlled individually on a large number of spots. This individual control for each spot is performed by the diamond micro reactor system. Individually addressable ejector elements in each reaction chamber allow the fabrication of individually sequenced oligonucleotides on practically every appropriately functionalized reaction substrate surface Žsilicon, polypropylene, glass, etc... Such oligonucleotides which are anchored on the reaction substrate, confined to small spots and arranged as an array are also called ‘DNA-chips’. DNA-chips are presently used in the development of new drugs, the evaluation of gene functions, the diagnostics of diseases and in other medical and biological applications. The fabrication of DNA-chips is currently done either by a process similar to photolithography w30,31x or by anchoring prefabricated DNA or oligonucleotides on a solid substrate,
Fig. 5. Technology.
M. Adamschik et al. r Diamond and Related Materials 10 (2001) 722᎐730
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Fig. 6. Ža. Array of 10 = 12 heaters, metallization; capillary wall system ŽSEM micrograph.; Žb. enlarged part of one micro heater.
which may be polypropylene w32x, etc. Both methods are relatively inflexible in respect to custom design. To illustrate the details involved, in the following the main steps of the oligonucleotide synthesis cycle will be shortly described.
4. Oligonucleotide synthesis DNA is a linear polymer consisting of a sugar᎐phosphate backbone and four different bases attached to the sugar moieties ŽFig. 9a.. The succession Žsequence. of the bases encodes the information. Synthesis of oligonucleotides Žshort parts of DNA chain, see Fig. 9b. is done by successive addition of sugar᎐phosphate᎐base units Žadenine, guanine, cytosine or tymine. to a growing oligonucleotide chain anchored on a solid support. To increase the chain by one base unit a synthesis cycle with the following most important synthesis steps has to be carried out ŽFig. 10.: 1. Deprotection and neutralization. 2. Coupling Žof one base.. These two steps will be discussed in more detail in the following. The oligonucleotide chain is made chemically inaccessible by a temporary protection group ŽFig. 9b, Fig. 11a.. This group is removed by an acid
Fig. 7. Ža. Nozzle plate ᎏ reaction chamber unit, column of 10 reaction chambers and 10 = 2 nozzles; Žb. SEM micrograph of nozzle.
Fig. 8. Ža. Micro reaction device; Žb. top view into one reaction chamber; Žc. complete chip with electrical leads for addressing the heaters.
ŽFig. 11b. followed by a neutralization with a basic solution ŽFig. 11c.. Thus, chemically reactive functionality is formed at the end of the oligonucleotide chain. To this functional site the next sugar᎐phosphate᎐base unit is coupled in the next step ŽFig. 11d.. To perform the coupling in the desired manner, the sugar᎐phosphate᎐base unit has to carry the temporary protection group to prevent reaction with itself and to prevent more than one coupling. To increase the oligonucleotide by one further base ŽFig. 11e. step Ža. and Žb.
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M. Adamschik et al. r Diamond and Related Materials 10 (2001) 722᎐730
Fig. 9. Ža. DNA-chain; Žb. oligonucleotide.
have to be repeated again Žtogether with other additional steps.. The step, where the spots on the reaction substrate are individually addressed, are the deprotection and neutralization step ŽFig. 11b,c.. This is performed by the diamond micro reactor system, which provides and injects the necessary aggressive chemicals Žacid and base.. A reaction Žsynthesis . substrate made of polypropylene represents the bottom of the reaction chamber and contains the anchored oligonucleotides. During the deprotection step the micro dosage unit and the reaction substrate are pressed together to form the chamber for the confined fluid and are disassembled after the reaction took place. The neutralization ŽFig. 11c. is necessary to prevent unwanted deprotection due to residual acid on the reaction substrate during the rinse in the sugar᎐phosphate᎐base solution ŽFig. 11d. which contains one sugar᎐base pair Žeither adenine, guanine, thiamine or cytosine.. This means
Fig. 10. Synthesis cycle.
that after neutralization the micro dosage unit is removed from the reaction substrate and the substrate is rinsed in a sugar᎐phosphate᎐base solution ŽFig. 11d. to increase the deprotected oligonucleotides by one base.
5. Performance of the technological building blocks of the micro dosage unit To verify the functionality of the micro dosage unit we first visualized the bubble nucleation of the micro heater elements Žarea s 30 = 30 m. in the ‘open mode’ Žwithout capillary wall system and nozzle plate. applying the pseudo-cinematographic visualization method. Accordingly in this investigation the measurement has been performed with the reaction fluids used in the synthesis cycle. Here collidine, a basic solution, and
Fig. 11. Synthesis with diamond micro reaction system: Ža. oligonucleotide before synthesis cycle with three bases; Žb. acid injection and removal of protecting group; Žc. neutralization with base; Žd. coupling of one base by rinsing in nucleotide solution; Že. oligonucleotide after synthesis cycle with four bases.
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Fig. 12. Pseudo cinematographic recording of nucleation in ‘open mode’ of collidine: Pel s 2.3 W, t pulse s 5 s, f repetion s 3 kHz.
Fig. 13. Pseudo cinematographic recording of nucleation in ‘open mode’ of TCA: Pel s 2.3 W, t pulse s 5 s, f repetion s 3 kHz.
trichloro acetic acid ŽTCA., both solved in an organic solution are applied for the deprotection and neutralization step. In the open mode all relevant properties like dynamic bubble nucleation, the necessary electrical power for nucleation, chemical or mechanical attack of the heater surface or the metallization can be investigated and extracted. Nucleation with water and ink have already been published previously w18x. Fig. 12 shows the bubble nucleation of collidine. Due to the high temperature stability of diamond the power density of 2.6 GWrm2 could be applied on 30 = 30 m heater elements far below thermal breakdown resulting in a very fast nucleation time of approximately 1.5 s. The maximum nucleation frequency was limited by the average thermal heating of the substrate to approximately 40 kHz. No mechanical or chemical damage of the diamond heater surface or the metallization could be detected showing that indeed no passivation layer is necessary on top of the diamond heater. Fig. 13 shows a comparable experiment with TCA.
Here again, no corrosion or other chemical or mechanical damage of the heater element and metallization could be observed. After approximately 3 s the bubble collapse begins for both liquids. To measure and visualize the drop ejection for a closed ejection system a real time cinematographic recording set-up has been used. The reaction fluid in this case was an organic solvent resulting in drop ejections as shown in Fig. 14. The liquid amount per drop can be estimated to approximately 30 pl allowing very precise control of the ejected liquid and thus mixing ratios. To prove the functionality of the system and to investigate the drop impact and the mixing behavior of two drops onto the reaction substrate a dual drop ejection experiment onto a transparent substrate has been performed. A pulse sequence has been applied onto the two heater elements resulting in a successively increasing amount of liquid onto the reaction substrate as shown in Fig. 15. For the first 10 drops the drops still
Fig. 14. Real time cinematographic recording of drop ejection; medium: organic solvent w19x.
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Fig. 15. Dual drop impact onto a transparent reaction substrate.
remain separated flowing together after approximately 20 drops corresponding to a volume of approximately 1 nl. This experiment has been successfully performed with water, 1-2-propanol, collidine and TCA, however, due to the best optical contrast water was used in the experiment shown in Fig. 15. In the oligonucleotide synthesis the reaction fluids show different wetting behavior on the surface due to different surface tensions. This is one reason, why in this case polypropylene Žnot transparent . has been selected. Good mixing of the fluids and good confinement of the fluids onto the reaction substrate could be observed. To validate the chemistry in the micro system and to investigate sealing between the bottom of the reaction chambers and the reaction substrate, a micro fluidic capillary system in the geometry of a meander instead of the diamond micro reactor system with square reaction chambers has been pressed onto a planar polypropylene substrate. The meander was connected with external tubes and successively provided with all necessary fluids for the chemical synthesis of oligonucleotides. Sealing could be obtained by proper surface termination of both surfaces and exploiting capillary effects. Fig. 16 shows the result of the synthesis. Good confinement even without substrate patterning is seen. The area of synthesized oligonucleotides can be identified after attaching a color marker to the oligonucleotides.
6. Conclusion A novel versatile diamond micro reaction system for
chemical reactions has been presented. The system consists of a micro dosage unit based on the bubble-jet principle, integrated reaction chambers with open bottom, a reaction substrate and an overlay carrier for liquid and electrical connections. The system is chemically inert due to the application of CVD-diamond films on decisive locations like the micro heater elements, the nozzle plate and the reaction chamber surface. Each reaction chamber contains two ejection elements supplied by two different fluids allowing precise injection and mixing of the chemicals. After the chemical reaction the synthesis substrate may be taken off for inspection. The reaction chambers are arranged in an array and each heater element is selectively addressable. The performance of the system has been investigated by visualization of the bubble nucleation and drop ejection of an organic solvent, acidic and basic solution. Power densities up to 30 GWrm2 resulting in sub-microsecond nucleation times could be applied to the diamond heater elements without thermal breakdown. No chemical or mechanical attack on the heater surface could be observed. As an example, the synthesis of oligonucleotides Žpart of a DNA-chain. has been discussed. The diamond micro reaction system allows parallel and highly flexible fabrication of ‘custom-design’ oligonucleotide arrays ŽDNA-chips. by selective deprotection of oligonucleotides anchored on a reaction substrate. In the future it seems feasible to still complement the system with sensors to monitor and control the reaction. Such a sensor may be the recently proposed diamond based pH-sensor w33x, the ion-sensitive ISFET
Fig. 16. Polypropylene substrate with synthesized oligonucleotids.
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w34x or electrodes for cyclic electro voltammetry making the system a true ‘lab-on-chip’.
Acknowledgements We thank A. Kaiser and Y. Men of the University of Ulm for the support at the device fabrication and the measurements of the bubble nucleation in the ‘open mode’. We would also like to thank A. Hildebrandt for developing electronic components for driving the diamond micro heaters. We acknowledge the cooperation with P. Gluche and A. Floter ¨ for providing diamondon-Si substrates. This work is supported by the German BMBF and DECHEMA under the classification number 03D00693. References w1x M. Werner et al., Review on Diamond Based Piezoresistive Sensors, ISIE’98, Proceedings, pp. 147᎐152. w2x I. Taher et al., Piezoresistive microsensors using p-type CVD diamond films, Sensors Actuators A 45 Ž1994. 35᎐43. w3x J.L. Davidson et al., CVD Diamond for Components and Emitters, 7th International Conference on New Diamond Science and Technology, Book of Abstracts, No. 16.1, City University of Hong Kong, Hong Kong, 23᎐28 July 2000. w4x D.R. Wur, J.L. Davidson, W.P. Kang, D.L. Kinser, Polycrystalline diamond pressure sensor, J. Micromech. Syst. 4 Ž1995. 34᎐41. w5x E. Kohn, P. Gluche, M. Adamschik, Diamond MEMS ᎏ a new emerging technology, Diamond Relat. Mater. 8 Ž1999. 934᎐940. w6x G.S. Yang et al., Single-structure heater and temperature sensor using a p-type polycrystalline diamond resistor, IEEE Electron Dev. Lett. 17 Ž1996. 250᎐252. w7x M. Aslam, G.S. Yang, A. Masood, Boron-doped vapor-deposited diamond temperature microsensors, Sensors Actuators A 45 Ž1994. 131᎐137. w8x R.D. McKeag, R.B. Jackman, Diamond UV photodetectors: sensitivity and speed for visible blind applications, Diamond Relat. Mater. 7 Ž1998. 513᎐518. w9x M. Adamschik, S. Ertl, P. Schmid, E. Kohn, Electrostatic Diamond Micro Switch, Transducers ’99, 10 th Int. Conf. on Solid-State Sensors and Actuators, Digest of Technical Papers, vol. 2, 1999, pp. 1284᎐1287. w10x S. Ertl, M. Adamschik, P. Schmid, P. Gluche, A. Floter, E. ¨ Kohn, Surface Micromachined Microswitch, Proc. of 10 th Int. Conf. on Diamond, Diamond-Like Materials, Carbon Nanotubes, Nitrides & Silicon Carbide, Diamond 1999, Prague Hilton Atrium, Czech Republic, 12᎐17 September 1999, pp. 970᎐974. w11x T. Shibata, Y. Kitamoto, K. Unno, E. Makino, Micromachining of diamond film MEMS applications, J. Microelectromech. Syst. 9 Ž1. Ž2000. 47᎐51. w12x E. Kohn, M. Adamschik, P. Schmid, S. Ertl, A. Floter, Dia¨ mond Electro-Mechanical Micro Devices ᎏ Technology and Performance, 7th International Conference on New Diamond Science and Technology, Book of Abstracts, City University of Hong Kong, Hong Kong, 23᎐28 July 2000, p. 3 w13x P. Schmid, M. Adamschik, S. Ertl, P. Gluche, E. Kohn, Modeling approach for CVD-Diamond based mechanical structures,
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Diamond micro system for bio-chemistry M. Adamschik a,U , M. Hinz c , C. Maier b, P. Schmida , H. Seliger c , E.P. Hofer b, E. Kohna a
b
Department of Electron De¨ ices and Circuits, Uni¨ ersity of Ulm, D-89081 Ulm, Germany Department of Measurement Control and Microtechnology, Uni¨ ersity of Ulm, Control and Microtechnology, D-89081 Ulm, Germany c Department of Polymers, Uni¨ ersity of Ulm, D-89081 Ulm, Germany
Abstract This work illustrates the potential of diamond micro system technologies progressively developed in the last years for an application in the bio-chemical field. A diamond micro reactor system based on a novel integration concept is presented and the role of diamond in this generic system is described. It consists of reaction chambers with removable bottom and integrated micro dosage elements allowing the ejection and mixture of two different fluids onto the removable bottom substrate. As an example, this system is used in a novel DNA-synthesis cycle. In this application the diamond micro reactor system combined with a specifically designed chemistry for the DNA-synthesis enables the parallel production of DNA-chain-clusters with individual sequence arranged in an array ŽDNA-Chip.. 䊚 2001 Elsevier Science B.V. All rights reserved. Keywords: Diamond micro system; Bio-chemistry; DNA; Diamond MEMS; Diamond actuators
1. Introduction Many diamond-based sensor and actuator structures like piezoresistive- w1,2x, pressure- w3,4x, accelerationw5x, temperature- w6,7x, UV-sensors w8x, micro switches w9,10x and various micro electromechanical system ŽMEMS. applications w11᎐13x have been reported previously. Beyond these applications and the application as electrochemical electrode w14᎐17x in the present work the potential of diamond for devices in the new field of bio-chemistry and nano-chemistry shall be demonstrated. The role of diamond in a generic chemical micro reactor system w19x will be described. Chemical micro reactors have emerged as an essential part of all bio-chemical synthesis and analysis systems w20᎐22x.
U
Corresponding author. University of Ulm, Albert-Einstein-Allee 45, D-89081 Ulm, Germany. Tel.: q49-731-502-6179; fax: q49-731502-6155. E-mail address: [email protected] ŽM. Adamschik..
The system presented here consists of three main parts ŽFig. 1.: 1. an overlay carrier for electrical and liquid supply; 2. a micro dosage unit based on the bubble jet principle combined with an integrated array of reaction chambers with open bottom; and 3. a removable substrate representing the bottom of the reaction chamber array. Integrated into the micro dosage unit are individually controllable diamond bubble-jet elements for precise injection of two different fluids into the reaction chambers. The diamond bubble-jet element has in essence already been published previously w5,18,23᎐25x and is shown in Fig. 2. This element combines high robustness, good chemical inertness and simple technology, thus serving as a reliable work horse for integration into such a system. The bubble-jet element had already been character-
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M. Adamschik et al. r Diamond and Related Materials 10 (2001) 722᎐730
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Fig. 1. Schematic cross-section of micro reactor system.
ized extensively, showing its outstanding properties w27,28x. Due to the high hardness, high mechanical strength and chemical inertness no passivation layers are necessary on top of the diamond heater element to prevent cavitation damages or chemical attack of the heater surface resulting in high reliability, low thermal induced stresses, small heat losses, and a simple technology. The low specific heat capacity allows fast temperature changes from room temperature up to the spinodalean limit Ž312⬚C for water w24x. necessary for proper overheating and bubble nucleation of the fluids. The good electrical isolating properties of undoped diamond combined with the high thermal conductivity allows electrically insulating layers w29x on top of the heater surface to prevent leakage currents between individual heaters during the ejection of acidic or basic solutions. From lifetime tests over 10 8 nucleation cycles could be extracted without deterioration showing that the heater element itself is not limiting the lifetime w25x. Due to the material properties the diamond heaters are able to withstand power densities up to 30 GWrm2 w25x being 10 times that of the nucleation power density in a normal operation. Fig. 3 shows an example of the electrical power applied onto a 60 = 60m heater element vs. minimum heating time for nucleation using a water covered heater element. Here the maximum power of 30 GWrm2 has been applied, resulting in an ultra-fast sub-microsecond nucleation time. Fig. 4 shows the time for damage for this extremely high power level, which is approximately four times of that necessary for nucleation. The new integrated diamond micro reactor system discussed in the following contains two bubble-jet elements in a single reaction chamber with a heater area of 30 = 30 m to overheat the liquids ŽFig. 5.. The reaction chambers are arranged as an array. The micro dosage unit is bonded into an overlay carrier, which contains the liquid and electrical connections. The open bottom of the reaction chamber array can be pressed
onto the surface of a planar or patterned reaction substrate, which can be removed for inspection after the chemical reaction took place. The amount of ejected liquid can be adjusted very precisely by sequentially driving the heater elements, resulting in a digital increase by 30-pl increments per drop w24x. Thus, the system enables tight control of chemical exotherm or endotherm reactions, mixing, etc. Due to the chemical inertness, the parallel arrangement of the reaction chambers, the selectively addressable heater elements and the precisely adjustable volumes of ejected liquids, this diamond chip represents a generic building block for nano-chemical systems. As a first example the synthesis of oligonucleotides Žoligonucleotide s part of a DNA-chain, see Fig. 9b. will be presented and discussed. The system allows fast and parallel fabrication of oligonucleotides with individual sequences Ž‘custom design’. on the surface of a reaction substrate by combining a specially designed chemistry w26x. Due to the aggressive nature of the chemistry all major parts of the system need to be chemically inert.
Fig. 2. Diamond bubble-jet ejector element.
Fig. 3. Applied electrical heating power vs. nucleation time w25x.
2. Technology The heart of the system is the micro dosage unit ŽFig. 1, part II. consisting of an array of open bottom reaction chambers with integrated bubble-jet injectors. It is fabricated from two parts as illustrated in Fig. 5a,b
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Fig. 4. Applied electrical heating power vs. time until destruction w25x.
using two different diamond-on-Si wafers. The assembled part Žpart 1 and 2 in Fig. 5. is then mounted into the fluidic and electrical supply carrier. Each micro reaction chamber contains two ‘bubblejet’ injector elements as indicated above, based on highly doped diamond micro heater elements ŽFig. 5a.. The diamond heater elements are grown by microwave plasma CVD onto a thermal insulation layer and are doped by boron. After the growth step dry etching was used for patterning. For the supply and the distribution of the reaction fluids on this part a photosensitive and chemical resistant polyimide capillary wall system was used, because this part of the device is not thermally stressed. The second part consists of the diamond nozzle plate and reaction chambers ŽFig. 5b.. The rectangular reaction chamber geometry was formed by anisotropic silicon etching using KOH. In the etching process diamond again offers the advantage of a selective etch stop. A 5-m thick CVD-diamond film was grown by PECVD and holes with a diameter of 30 m were etched by dry etching. In the last step the nozzle plate ᎏ reaction chamber part is bonded onto the top of the capillary wall system of part 1 forming a leak-tight microfluidic system. To connect the micro fluidic capillary system with conventional tubes an overlay carrier was fabricated into which the micro reaction chip was
bonded. It contains an integrated fluidic system, fluidic connectors and electrical connectors. Fig. 5c represents a schematic view of a single reaction chamber of the complete array. Fig. 6a shows part 1 of the device and a detailed view of one heater element. Fig. 7 shows part 2, the nozzle plate with integrated reaction chambers and a detailed SEM micrograph of a single nozzle. Anisotropic and smooth sidewalls can be seen, preventing vortexes during drop ejection. Fig. 8a shows a device with 10 reaction chambers in a one-dimensional array after chip bonding of part 1 and 2. Fig. 8b depicts a top view into a single reaction chamber showing the nozzles, the metallization and the diamond heater elements through the transparent CVD-diamond nozzle plate. The completely assembled system with the high-grade steel carrier and its electrical connections can be seen in Fig. 8c.
3. Application As the first application the synthesis of oligonucleotides, which are a segment of a DNA-chain, is discussed. Such a synthesis is carried out in a cycle of four steps. The synthesis is supposed to be controlled individually on a large number of spots. This individual control for each spot is performed by the diamond micro reactor system. Individually addressable ejector elements in each reaction chamber allow the fabrication of individually sequenced oligonucleotides on practically every appropriately functionalized reaction substrate surface Žsilicon, polypropylene, glass, etc... Such oligonucleotides which are anchored on the reaction substrate, confined to small spots and arranged as an array are also called ‘DNA-chips’. DNA-chips are presently used in the development of new drugs, the evaluation of gene functions, the diagnostics of diseases and in other medical and biological applications. The fabrication of DNA-chips is currently done either by a process similar to photolithography w30,31x or by anchoring prefabricated DNA or oligonucleotides on a solid substrate,
Fig. 5. Technology.
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Fig. 6. Ža. Array of 10 = 12 heaters, metallization; capillary wall system ŽSEM micrograph.; Žb. enlarged part of one micro heater.
which may be polypropylene w32x, etc. Both methods are relatively inflexible in respect to custom design. To illustrate the details involved, in the following the main steps of the oligonucleotide synthesis cycle will be shortly described.
4. Oligonucleotide synthesis DNA is a linear polymer consisting of a sugar᎐phosphate backbone and four different bases attached to the sugar moieties ŽFig. 9a.. The succession Žsequence. of the bases encodes the information. Synthesis of oligonucleotides Žshort parts of DNA chain, see Fig. 9b. is done by successive addition of sugar᎐phosphate᎐base units Žadenine, guanine, cytosine or tymine. to a growing oligonucleotide chain anchored on a solid support. To increase the chain by one base unit a synthesis cycle with the following most important synthesis steps has to be carried out ŽFig. 10.: 1. Deprotection and neutralization. 2. Coupling Žof one base.. These two steps will be discussed in more detail in the following. The oligonucleotide chain is made chemically inaccessible by a temporary protection group ŽFig. 9b, Fig. 11a.. This group is removed by an acid
Fig. 7. Ža. Nozzle plate ᎏ reaction chamber unit, column of 10 reaction chambers and 10 = 2 nozzles; Žb. SEM micrograph of nozzle.
Fig. 8. Ža. Micro reaction device; Žb. top view into one reaction chamber; Žc. complete chip with electrical leads for addressing the heaters.
ŽFig. 11b. followed by a neutralization with a basic solution ŽFig. 11c.. Thus, chemically reactive functionality is formed at the end of the oligonucleotide chain. To this functional site the next sugar᎐phosphate᎐base unit is coupled in the next step ŽFig. 11d.. To perform the coupling in the desired manner, the sugar᎐phosphate᎐base unit has to carry the temporary protection group to prevent reaction with itself and to prevent more than one coupling. To increase the oligonucleotide by one further base ŽFig. 11e. step Ža. and Žb.
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Fig. 9. Ža. DNA-chain; Žb. oligonucleotide.
have to be repeated again Žtogether with other additional steps.. The step, where the spots on the reaction substrate are individually addressed, are the deprotection and neutralization step ŽFig. 11b,c.. This is performed by the diamond micro reactor system, which provides and injects the necessary aggressive chemicals Žacid and base.. A reaction Žsynthesis . substrate made of polypropylene represents the bottom of the reaction chamber and contains the anchored oligonucleotides. During the deprotection step the micro dosage unit and the reaction substrate are pressed together to form the chamber for the confined fluid and are disassembled after the reaction took place. The neutralization ŽFig. 11c. is necessary to prevent unwanted deprotection due to residual acid on the reaction substrate during the rinse in the sugar᎐phosphate᎐base solution ŽFig. 11d. which contains one sugar᎐base pair Žeither adenine, guanine, thiamine or cytosine.. This means
Fig. 10. Synthesis cycle.
that after neutralization the micro dosage unit is removed from the reaction substrate and the substrate is rinsed in a sugar᎐phosphate᎐base solution ŽFig. 11d. to increase the deprotected oligonucleotides by one base.
5. Performance of the technological building blocks of the micro dosage unit To verify the functionality of the micro dosage unit we first visualized the bubble nucleation of the micro heater elements Žarea s 30 = 30 m. in the ‘open mode’ Žwithout capillary wall system and nozzle plate. applying the pseudo-cinematographic visualization method. Accordingly in this investigation the measurement has been performed with the reaction fluids used in the synthesis cycle. Here collidine, a basic solution, and
Fig. 11. Synthesis with diamond micro reaction system: Ža. oligonucleotide before synthesis cycle with three bases; Žb. acid injection and removal of protecting group; Žc. neutralization with base; Žd. coupling of one base by rinsing in nucleotide solution; Že. oligonucleotide after synthesis cycle with four bases.
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Fig. 12. Pseudo cinematographic recording of nucleation in ‘open mode’ of collidine: Pel s 2.3 W, t pulse s 5 s, f repetion s 3 kHz.
Fig. 13. Pseudo cinematographic recording of nucleation in ‘open mode’ of TCA: Pel s 2.3 W, t pulse s 5 s, f repetion s 3 kHz.
trichloro acetic acid ŽTCA., both solved in an organic solution are applied for the deprotection and neutralization step. In the open mode all relevant properties like dynamic bubble nucleation, the necessary electrical power for nucleation, chemical or mechanical attack of the heater surface or the metallization can be investigated and extracted. Nucleation with water and ink have already been published previously w18x. Fig. 12 shows the bubble nucleation of collidine. Due to the high temperature stability of diamond the power density of 2.6 GWrm2 could be applied on 30 = 30 m heater elements far below thermal breakdown resulting in a very fast nucleation time of approximately 1.5 s. The maximum nucleation frequency was limited by the average thermal heating of the substrate to approximately 40 kHz. No mechanical or chemical damage of the diamond heater surface or the metallization could be detected showing that indeed no passivation layer is necessary on top of the diamond heater. Fig. 13 shows a comparable experiment with TCA.
Here again, no corrosion or other chemical or mechanical damage of the heater element and metallization could be observed. After approximately 3 s the bubble collapse begins for both liquids. To measure and visualize the drop ejection for a closed ejection system a real time cinematographic recording set-up has been used. The reaction fluid in this case was an organic solvent resulting in drop ejections as shown in Fig. 14. The liquid amount per drop can be estimated to approximately 30 pl allowing very precise control of the ejected liquid and thus mixing ratios. To prove the functionality of the system and to investigate the drop impact and the mixing behavior of two drops onto the reaction substrate a dual drop ejection experiment onto a transparent substrate has been performed. A pulse sequence has been applied onto the two heater elements resulting in a successively increasing amount of liquid onto the reaction substrate as shown in Fig. 15. For the first 10 drops the drops still
Fig. 14. Real time cinematographic recording of drop ejection; medium: organic solvent w19x.
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Fig. 15. Dual drop impact onto a transparent reaction substrate.
remain separated flowing together after approximately 20 drops corresponding to a volume of approximately 1 nl. This experiment has been successfully performed with water, 1-2-propanol, collidine and TCA, however, due to the best optical contrast water was used in the experiment shown in Fig. 15. In the oligonucleotide synthesis the reaction fluids show different wetting behavior on the surface due to different surface tensions. This is one reason, why in this case polypropylene Žnot transparent . has been selected. Good mixing of the fluids and good confinement of the fluids onto the reaction substrate could be observed. To validate the chemistry in the micro system and to investigate sealing between the bottom of the reaction chambers and the reaction substrate, a micro fluidic capillary system in the geometry of a meander instead of the diamond micro reactor system with square reaction chambers has been pressed onto a planar polypropylene substrate. The meander was connected with external tubes and successively provided with all necessary fluids for the chemical synthesis of oligonucleotides. Sealing could be obtained by proper surface termination of both surfaces and exploiting capillary effects. Fig. 16 shows the result of the synthesis. Good confinement even without substrate patterning is seen. The area of synthesized oligonucleotides can be identified after attaching a color marker to the oligonucleotides.
6. Conclusion A novel versatile diamond micro reaction system for
chemical reactions has been presented. The system consists of a micro dosage unit based on the bubble-jet principle, integrated reaction chambers with open bottom, a reaction substrate and an overlay carrier for liquid and electrical connections. The system is chemically inert due to the application of CVD-diamond films on decisive locations like the micro heater elements, the nozzle plate and the reaction chamber surface. Each reaction chamber contains two ejection elements supplied by two different fluids allowing precise injection and mixing of the chemicals. After the chemical reaction the synthesis substrate may be taken off for inspection. The reaction chambers are arranged in an array and each heater element is selectively addressable. The performance of the system has been investigated by visualization of the bubble nucleation and drop ejection of an organic solvent, acidic and basic solution. Power densities up to 30 GWrm2 resulting in sub-microsecond nucleation times could be applied to the diamond heater elements without thermal breakdown. No chemical or mechanical attack on the heater surface could be observed. As an example, the synthesis of oligonucleotides Žpart of a DNA-chain. has been discussed. The diamond micro reaction system allows parallel and highly flexible fabrication of ‘custom-design’ oligonucleotide arrays ŽDNA-chips. by selective deprotection of oligonucleotides anchored on a reaction substrate. In the future it seems feasible to still complement the system with sensors to monitor and control the reaction. Such a sensor may be the recently proposed diamond based pH-sensor w33x, the ion-sensitive ISFET
Fig. 16. Polypropylene substrate with synthesized oligonucleotids.
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w34x or electrodes for cyclic electro voltammetry making the system a true ‘lab-on-chip’.
Acknowledgements We thank A. Kaiser and Y. Men of the University of Ulm for the support at the device fabrication and the measurements of the bubble nucleation in the ‘open mode’. We would also like to thank A. Hildebrandt for developing electronic components for driving the diamond micro heaters. We acknowledge the cooperation with P. Gluche and A. Floter ¨ for providing diamondon-Si substrates. This work is supported by the German BMBF and DECHEMA under the classification number 03D00693. References w1x M. Werner et al., Review on Diamond Based Piezoresistive Sensors, ISIE’98, Proceedings, pp. 147᎐152. w2x I. Taher et al., Piezoresistive microsensors using p-type CVD diamond films, Sensors Actuators A 45 Ž1994. 35᎐43. w3x J.L. Davidson et al., CVD Diamond for Components and Emitters, 7th International Conference on New Diamond Science and Technology, Book of Abstracts, No. 16.1, City University of Hong Kong, Hong Kong, 23᎐28 July 2000. w4x D.R. Wur, J.L. Davidson, W.P. Kang, D.L. Kinser, Polycrystalline diamond pressure sensor, J. Micromech. Syst. 4 Ž1995. 34᎐41. w5x E. Kohn, P. Gluche, M. Adamschik, Diamond MEMS ᎏ a new emerging technology, Diamond Relat. Mater. 8 Ž1999. 934᎐940. w6x G.S. Yang et al., Single-structure heater and temperature sensor using a p-type polycrystalline diamond resistor, IEEE Electron Dev. Lett. 17 Ž1996. 250᎐252. w7x M. Aslam, G.S. Yang, A. Masood, Boron-doped vapor-deposited diamond temperature microsensors, Sensors Actuators A 45 Ž1994. 131᎐137. w8x R.D. McKeag, R.B. Jackman, Diamond UV photodetectors: sensitivity and speed for visible blind applications, Diamond Relat. Mater. 7 Ž1998. 513᎐518. w9x M. Adamschik, S. Ertl, P. Schmid, E. Kohn, Electrostatic Diamond Micro Switch, Transducers ’99, 10 th Int. Conf. on Solid-State Sensors and Actuators, Digest of Technical Papers, vol. 2, 1999, pp. 1284᎐1287. w10x S. Ertl, M. Adamschik, P. Schmid, P. Gluche, A. Floter, E. ¨ Kohn, Surface Micromachined Microswitch, Proc. of 10 th Int. Conf. on Diamond, Diamond-Like Materials, Carbon Nanotubes, Nitrides & Silicon Carbide, Diamond 1999, Prague Hilton Atrium, Czech Republic, 12᎐17 September 1999, pp. 970᎐974. w11x T. Shibata, Y. Kitamoto, K. Unno, E. Makino, Micromachining of diamond film MEMS applications, J. Microelectromech. Syst. 9 Ž1. Ž2000. 47᎐51. w12x E. Kohn, M. Adamschik, P. Schmid, S. Ertl, A. Floter, Dia¨ mond Electro-Mechanical Micro Devices ᎏ Technology and Performance, 7th International Conference on New Diamond Science and Technology, Book of Abstracts, City University of Hong Kong, Hong Kong, 23᎐28 July 2000, p. 3 w13x P. Schmid, M. Adamschik, S. Ertl, P. Gluche, E. Kohn, Modeling approach for CVD-Diamond based mechanical structures,
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