Italy, Salento, Lecce

1. Italy, Salento, Lecce

3. NNL- National technology laboratories

4. Hybrid systems for biosensing on chip V. Arima National Nanotechnology Laboratory of INFM-CNR HI-Tech. – District – ISUFI University of Lecce IIT- Italian Institute of Technology Via Arnesano 73100 LECCE – ITALY Valentina.arima@unile.it

5. Outline • Biosensing in nanotechnology • The electrical approach • Optical detection (Dr. P.P.Pompa)

6. Biosensing in nanotechnology Fast and reliable detection of substances important to human welfare is much-needed, both in medicine and in the food industry. To this end, biosensors play an important role. The sensors will typically represent a combination of biochemistry, electronics or optics and surface chemistry

7. Electrochemical biosensors Many leading devices for self-measurement of blood glucose are based on static electrochemistry. A fixed input signal (such as an applied voltage) from the blood glucose meter results in an output signal that correlates to the glucose concentration in the sample AgaMatrix device

8. inlets for the liquids • continuos flow of liquids from the inlets to the detection area through microchannels • PCR cycles during the flow outlet gold electrode sensor for temperature control Connection to PCB inlet heater Detection area Amplification area using PCR K. Sun et al. / Sensors and Actuators B 84 (2002) 283–289 DNA chips Chips for amplification and detection of DNA in organic fluids

9. Outline • Biosensing in nanotechnology • The electrical approach • Optical detection (Dr. P.P.Pompa)

10. Planar-nanotips - EBL Interdigited nanoelectrods photolithography Mesa structures - photolithography Geometry of devices

11. Surface chemistry Biomolecules interconnected to nanodevices: the azurin example • Azurin monolayers on silicon oxide • Azurins on gold Azurin mediates electron-transfer in the denitrifying chains of Pseudomonas aeruginosa • ET by means of a reversible redox reaction involving the copper site (two stable electronic configurations: Cu(I) and Cu(II)) • The disulfide bridge allows chemisorption

12. Azurin monolayers on silicon oxide NC-AFM images of WT Azurin immobilized on SiO2 functionalized by a self-assembled 3-MPTS monolayeremploying the disulfide bridge RMS increases from 0.3 to 3.17nm; lateral dimension (after tip deconvolution) around 4nm in agreement with crystallographic data G.Maruccio et al , Adv. Mat., 17, 816-822 (2005)

13. Azurins on gold 50nM sodium acetate, pH 4.6 (isoelectric point) at 4°C for 1.5h (a) Azurin immobilized on Au(111) for STM experiments (b) Three dimensional STM view showing azurin molecules adsorbed on Au (111) recorded in air. Bias voltage, -700mV, current setpoint, 1nA. Scan area, 130x130 nm2; vertical range, 0.6 nm. Scan rate 4Hz. (c) STM topographic image of azurin adsorbed on Au(111) acquired in constant-current mode in air. The proteins are clearly visible as bright spots. (d) Height profile of azurin imaged by STM. The lateral size (FWHM) around 4-6 nm is in good agreement with the reported crystallographic data. V. Frascerra et al, IEEE Transactions on Nanotechnology (T-NT) 4, 637-640 (2005)

14. Planar-nanotips G. Maruccio et al., Microelectronic Engineering 67-68, 838-844 (2003) • Typical open-circuit resistance greater than 1 TΩ (100fA at 6V) • High throughput (90%) • Transport experiments at the surface of a solid Pre-exposure technique

15. Planar devices Intermolecular transport by hopping • Left scale: I(V) characteristics of devices made with azurin variants for two different gate voltages. • Right scale: Simulated current-voltage characteristics (red curve) of the protein FET for a device containing 8 protein chains connecting the source and the drain (Vg=1V was assumed in the calculation) G.Maruccio et al , Adv. Mat., 17, 816-822 (2005)

16. Towards single-molecule devices Au-cyanide electroplating set-up • Bath temperature 55 oC, pH=4.3 • Reference electrode: 20 cm2 • Anode-Cathode Current: 70 mA • Anode-Cathode distance: 7 cm • Low throughput (20-30%) P. Visconti et al., Mater. Sci. Eng. C-Biomimetic Supramol. Syst. 23 (6-8), 889-892 (2003). P. Visconti et al., Nanotechnology 15 (7), 807-811 (2004).

17. Reduction of leakage current • All processes at wafer scale • Low-cost mass production of nanoscale circuits Al-rich layers converted into a stable native oxide in a home-made oven after the cap layer removal continuous flow of water steam carried by N2 gas G.Maruccio et al. , Small (in press)

18. Mesa nanostructures • Dominant features: maxima in the range ±2.2 V ÷ ±3.4 V • NDR due to resonant tunneling resulting from the alignment of two narrow energy states localized on Cu(II) and in the region of RSSR bridge • Molecular features gradually disappear after a couples of weeks G.Maruccio et al , Small (in press)

19. gel Voltammograms of native azurin and its derivatives onto interdigited Cr/Au electrodes (the reference electrode was Au tip coated with silver or saturated calomel) Solution Interdigited electrods: towards an azurin-based biosensor

20. Electrical detection in DNA chips C.-Y. Tsai et al. Microsystem Technologies 11 (2005) 91–96

21. PCR chips PCR (Polymerase Chain Reaction) is a chemical amplification reaction of DNA. PCR is a highly specific reaction, capable of creating large amounts of copied DNA fragments from minute amounts of samples • Micro-chamber for PCR in PDMS • Nano-heaters electrodes

22. Acknowledgements Giuseppe Maruccio Antonio Della Torre Alessandro Bramanti Elisabetta Primiceri Eliana D’Amone Diego Mangiullo Stefania Sabella Vanessa Frascerra Adriana Biasco Paolo Visconti Pasquale Marzo Roman Krahne Ross Rinaldi Franco Calabi Roberto Cingolani The authors gratefully acknowledge the support by the SAMBA (Self-Assembling of copper Metalloproteins at nanoscale for Biodevice Applications) European project, the IIT and the Italian MIUR (FIRB Molecular Nanodevices)