A NOVEL APPROACH TO MAGNETIC A NOVEL APPROACH TO MAGNETIC FIELD BIOSENSORS: NMR AND SQUID FIELD BIOSENSORS: NMR AND SQUID

DETECTIONDETECTION

A. Valsesiaa, P. Colpoa, F. Rossia, P. Arosiob, M. Marianic,d, M. Cortic, d, M.F. Casulae, A. Lascialfarib,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

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-Fe2O3 superparamagnetic nanoparticles [2]

IgG

BSA

AbIgG-c-Biotin

Streptavidin

Fe2O3-c-PMA-c-Biotin

ppAA

Magnetization Measurements

Magnetic data depend on the concentration of Ab-IgG molecules blocked on the surface. The magnetic moment increasesThe magnetic moment increases in presence of γ-Fe2O3 with respect to the substrate +(protein) probe. This represents the represents the method of detectionmethod of detection on which MFB ( biochipsbiochips ! ! ) are based.

! Specific biological recognition !! Specific biological recognition !

Ch1 Ch2 Ch3 Ch40

2

4

6

8

10

12

14

16

De

ltaF

(Hz)

IgG AbIgG Strept Fe2O3

AbIgG=30 AbIgG=50 AbIgG=100 AbIgG=0

Ch1 Ch2 Ch3 Ch40

2

4

6

8

10

12

14

16

De

ltaF

(Hz)

IgG AbIgG Strept Fe2O3

AbIgG=30 AbIgG=50 AbIgG=100 AbIgG=0

QCM testQCM test SQUIDSQUID

0 10 20 30 400.0

0.1

0.2

0.3

0.4

0.5

hydrodynamic diameter = 270 nm

mw(1

0^-

6em

u/m

g)

AbIgG-biotin concentration

H = 500G

0 10 20 30 40 50 60 70 80 90 1000.0

0.1

0.2

0.3

0.4

hydrodynamic diameter = 200 nm

mw

(10

^-6

em

u/m

g)

AbIgG-biotin concentration

H = 500G

QCM measurements:

•Frequency shift as a function of the biotinated Ab-IgG molecules concentration

SQUID measurements:

* Room temperature

* Constant magnetic field H = 500 Oe

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-Fe2O3 superparamagnetic nanoparticles with mild shaking

• Diffusion of streptavidin with mild shaking

* Room temperature* 1H-NMR relaxation times T1 and T2 evaluated at 20.1 and 41 MHz as a function of time after the addition of streptavidin in Agarose gel where biotinated modified γ-Fe2O3 superparamagnetic nanoparticles were included

• Different relaxation times adding streptavidin with respect to np-hydrogel => Sensitivity of NMRSensitivity of NMR

The longitudinal relaxation time T1 is weakly influenced by addition of streptavidin to gel with biotinated modified γ-Fe2O3 but ………..

a 10-15% change of the transverse relaxation time T2 method of detection of probe-analyte interactionmethod of detection of probe-analyte interaction

Agarose

Streptavidin

Fe2O3-c-PMA-c-Biotin

1H-NMR relaxation times of the Agarose gel• at 20.1 MHz: T1 = 2.92 sec.; T2 = 98 msec.• at 41 MHz: T1 = 2.90 sec.; T2 = 103 msec.

[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 remarksTwo 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 ( BIOCHIPSBIOCHIPS ! ), the specific biological recognition biotin-streptavidinspecific biological recognition biotin-streptavidin was obtained by means by means of SQUID magnetic measurementsof SQUID magnetic measurements. Very interesting perspectives using 11H-NMR detection technique on “bulk” H-NMR detection technique on “bulk” probesprobes were presented.

AcknowledgmentsAcknowledgments

Fondazione Cariplo is gratefully acknowledged for having funded the project