1. A NOVEL APPROACH TO MAGNETIC FIELD BIOSENSORS: NMR AND SQUID DETECTION A. Valsesia a, P. Colpo a, F. Rossi a, P. Arosio b, M. Mariani c,d, M. Corti c, d, M.F. Casula e, A. Lascialfari b,c, d a European Commission, Joint Research Center, IHCP, Ispra (VA), Italy b Department of Molecular Sciences Applied to Biosystems - DISMAB, Università degli Studi di Milano, Milano, Italy c Department of Physics Volta, University of Pavia, Pavia, Italy d S3-CNR-INFM, Modena, Italy e Dipartimento di Scienze Chimiche, Università di Cagliari, Cagliari, Italy

2. We have studied novel approaches for the realization of Magnetic Field Effect Biosensors (MFBs), by optimizing the technique of immobilization of biomolecular probes on the surface and in the bulk. By using maghemite nanoparticles for marking the biomolecules, we obtained a good sensitivity of the detection method of MFBs using Nuclear Magnetic Resonance (NMR) and SQUID. Introduction MFBs: Approach 1 Plasma Deposited Poly Acrylic Acid (ppAA) [1] Adsorption of human IgG Blocking of the unreacted surface groups by BSA Reaction with biotinated Ab-IgG molecules at different concentrations Absorption of streptavidin Absorption of biotinated modified γ-Fe 2 O 3 superparamagnetic nanoparticles [2] IgG BSA AbIgG-c-Biotin Streptavidin Fe 2 O 3 -c-PMA-c-Biotin ppAA

3. Magnetization Measurements The magnetic moment increases represents the method of detectionbiochips ! Magnetic data depend on the concentration of Ab-IgG molecules blocked on the surface. The magnetic moment increases in presence of γ-Fe 2 O 3 with respect to the substrate +(protein) probe. This represents the method of detection on which MFB ( biochips ! ) are based. ! Specific biological recognition ! QCM test SQUID QCM measurements: Frequency shift as a function of the biotinated Ab-IgG molecules concentration SQUID measurements: * Room temperature * Constant magnetic field H = 500 Oe

4. NMR Measurements (preliminary results) MFBs: Approach 2 Hydrogel of Agarose (1%) directly prepared in the glass tube for NMR measurements Diffusion of biotinated modified γ-Fe 2 O 3 superparamagnetic nanoparticles with mild shaking Diffusion of streptavidin with mild shaking * Room temperature * 1 H-NMR relaxation times T 1 and T 2 evaluated at 20.1 and 41 MHz as a function of time after the addition of streptavidin in Agarose gel where biotinated modified γ-Fe 2 O 3 superparamagnetic nanoparticles were included Different relaxation times adding streptavidin with respect to np-hydrogel => Sensitivity of NMR The longitudinal relaxation time T 1 is weakly influenced by addition of streptavidin to gel with biotinated modified γ-Fe 2 O 3 but ……….. method of detection of probe-analyte interaction a 10-15% change of the transverse relaxation time T 2 method of detection of probe-analyte interaction Agarose Streptavidin Fe 2 O 3 -c-PMA-c-Biotin 1 H-NMR relaxation times of the Agarose gel at 20.1 MHz: T 1 = 2.92 sec.; T 2 = 98 msec. at 41 MHz: T 1 = 2.90 sec.; T 2 = 103 msec.

5. [1] F. Bretagnol, A. Valsesia, G. Ceccone, P. Colpo, D. Gilliland, L. Ceriotti, M. Hasiwa, and F. Rossi Plasma Processes and Polymers 3, 443 (2006). [2] C. J. Lin, R. A. Sperling, J. K. Li, T. Yang, P. Li, M. Zanella, W. H. Chang, and W. J. Parak Small 4, 334 (2008). Concluding remarks BIOCHIPSspecific biological recognition biotin-streptavidinby means of SQUID magnetic measurements 1 H-NMR detection technique on bulk probes Two different novel approaches for the realization of Magnetic Field Effect Biosensors (MFBs) based on SQUID magnetometry and Nuclear Magnetic Resonance detection were developed. Immobilizing biomolecular probe on functionalized surface ( BIOCHIPS ! ), the specific biological recognition biotin-streptavidin was obtained by means of SQUID magnetic measurements. Very interesting perspectives using 1 H-NMR detection technique on bulk probes were presented. Acknowledgments Fondazione Cariplo is gratefully acknowledged for having funded the project