A. Bongrain 1, H. Uetsuka 3, G. Lissorgues 2, E. Scorsone 1, L. Rousseau 2, L. Valbin 2, S. Saada 1, C. Gesset 1, P. Bergonzo 1 1 CEA, LIST, Laboratoire Capteur Diamant, GIF-SUR-YVETTE, F-91191, France. 2 ESIEE – ESYCOM Université Paris Est, Cité Descartes, BP99, Noisy Le Grand, France. 3 Diamond Research Center, AIST, Tsukuba, , Japan Measurement of DNA hybridization based on diamond micro-cantilever sensing I. Introduction MEMS structures can offer a real improvement for bio-detection applications in term of cost, miniaturization and sensitivity Diamond exhibits advantageous properties for MEMS-based bio-sensing applications -mechanical properties (High Young modulus, High resistance to fracture) -chemical properties (bio-chemical inert, covalent bonding using carbon chemistry) II. Diamond MEMS fabrication process IV. Cantilever’s resonance frequency measuring set up III. DNA grafting process on boron doped diamond cantilevers V. Results and discussion Detector and demodulator Piezo-electric cell driven voltage (frequency scan) Readout signal Spectrum analyser Laser beam Piezo-electric cell DNA attachment on boron doped diamond cantilevers Conclusion and perspectives Optimization of diamond nano-particules plasma etching duration optimization (step 4) Diamond nano-particules etching duration (min) Nano diamond residual density (cm -2 ) 10 µm O 2 /Ar Metal hard mask deposition on an oxidized Si substrate Moulds etching by DRIE* and metal removing Diamond nano- particules spreading Metal hard mask deposition inside the moulds and diamond nano-particules etching Metal hard mask removing and Diamond growth (MPCVD) Metal tracks deposition Structures releasing Back side DRIE Gas injection (H 2, CH 4 ) Gas extraction Microwave guide Microwave plasma Substrate support Polycrystalline diamond synthesized by MPCVD (Microwave Plasma Chemical Vapor Deposition) Ionization of a H 2 /CH 4 gas mixture (99:1) by microwave CH 4 supplies carbon atoms H 2 prevent from graphitic carbon growth MPCVD growth reactor for diamond growth on large surface (until 4 inches) SiO2 chemical etching and short BDD* growth 100 µm 4 inches 1)2) 3)4) 5)6) 7)8) - Versatile process - Adapted for large surface - No diamond etching or polishing required *BDD: Boron Doped Diamond *DRIE: Deep Reactive Ion Etching Cyclic voltammetry Chemical reaction Hydrogen or oxygen terminated surface of boron doped diamond Diazonium salt electrochemical reduction Electrochemical reduction of –NO 2 Attachment of cross-linker and thiol modified ss-DNA grafting Hybridization with complementary target DNA functionalized with a color maker Cyclic voltammetry Fluorescent tag Cyclic voltametry of NO2 to NH2 reduction in KCL 0.1M in DI water:ethanol (9:1) First cycle Four-step electrochemical DNA grafting process Attachment of 32-base DNA Before denaturation After denaturation Reference Measuring 97.45% 99.74% 100% Relative variation of the resonance frequency (%) 2.3 % 7 mm Liquid cell - Resonance frequency measured by Doppler laser interferometry - Cantilevers actuated by an external piezo-electric cell - Measurements in liquid 2 identical cantilevers on the chip 1 reference (bare cantilever) 1 measuring (functionalized) a- DNA grafting success checked by fluorescence after hybridization Fluorescence represented by the green light indicates that attached DNA is located on the measuring cantilever, only. Significant contrast between grafted area and non grafted area. b- Comparison of cantilever’s resonance frequency between before and after denaturation 1. First measurements in a Phosphate Buffer Solution (PBS) volume of 400 µL on both reference and measuring cantilevers, respectively. 2. Denaturation in NaOH 3. Second measurements in a purged PBS volume of 400 µL on both reference and measuring cantilevers, respectively. Significant decrease of the resonance frequency measured on the measuring cantilever after denaturation Same resonance frequency measured on the reference cantilever after denaturation Differential measurement of -75 Hz (2.3% of a 3 kHz-cantilever) c- Discussion Denaturation Decrease of electrostatic repulsions Hybridization Diamond and Biology C.E. Nebel et al, (2007). Measured resonance frequency shift mainly attributed to the surface stress contribution because: -The resonance frequency shift is negative and hence opposed that it should be if mass were the dominant contribution -Cantilever itself as a poor sensitivity to a mass change in liquid (cantilever length: 900 µm) -Calculations showed that the grafted DNA layer as an unsignificant effect on the overall cantilever Young modulus -DNA is negativelly charged (sugar phosphate) and hence electrostatic repulsions are induced between grated DNA strand -High cantilever sensitivity over surface stress change (~tens Hz/N/m) Detection of DNA denaturation by measuring cantilever’s resonance frequency shift Resonance frequency shift mainly attributed to a change of surface stress induced by electrostatic repulsons between grafted DNA Hybridization cycle Fluorescence intensity (arbitrary units) Yang et al.., Nature Materials, 1 (2002) 253 High structures sensitivity in dynamic mode Robust devices Stability of surface functionalization Transducers resistant to harsh chemical environment Cyclic voltametry of diazonium reduction on boron doped diamond Resonance frequency shift mainly attributed to a change of surface stress induced by electrostatic repulsons between grafted DNA Cantilevers sensitivity over surface stress (~tens Hz/N/m) offers large opportunities of bio-sensing applications (typical induced surface stress change ~tens mN/m)