Peptide-Nanotube Biochips for Label-Free Detection of Multiple Pathogens

Fabrication of the array of electrodes was carried out in a microfabrication clean room using common deposition and patterning techniques. Fabrication started with the deposition of a triple layer of Ti/Ni/Au (500Å /500Å /500Å ) on a 500-μm-thick Pyrex wafer. After the metal layers were patterned with standard photolithography and wet etching of the metals, a 1-μm-thick silicon oxide passivation coating was deposited and patterned. Finally, the passivation layer was removed from the pads and from the interdigitated area with a second photolithography step and oxide dry etching. The peptide nanotubes were prepared by self-assembly from the bolaamphiphilic peptide monomer bis(Na- amidoglycylglycine)-1,7-heptane dicarboxylate and used as templates for the immobilization of antibodies as reported previously.[19,20,9] To obtain antibody nanotubes, after centrifuging 1mL of the peptide-nanotube solution at 14 000 rpm for 1 h, the resulting pellet was suspended in 100μL of phosphate buffer (PB, 1mM, pH 7) containing either anti-E. coli (goat polyclonal, 0.1mgmL-1, Abcam), anti-S. typhi (rabbit polyclonal, 0.1mgmL-1, Abcam), or IgG from rabbit serum (0.1mgmL -1, Sigma) overnight. Then, 500mL of bovine serum albumin (BSA, 10mgmL-1) were added for 1 h to block free adsorption sites on the tubes and the nanotubes were washed once with PB by centrifugation. After mixing 10μL of antibody nanotubes with 10mL of bacteria solutions in PB with different concentrations for 1 h, 1μL of the resulting nanotube- bacteria adducts were spotted on each transducer and Z0 at 316 kHz was measured with a 1260 Solartron Impedance Analyzer by applying a peak-to-peak excitation voltage of 10mV. The x axis in Figure 3 shows the number of cells in the 10mL solution. In Figure 3b, Z0 at 15-s sedimentation time of bacteria-nanotube complexes was converted into a color code by calculating the percentage variation of this parameter with respect to the maximum signal and tuning the brightness of the spot accordingly. Scanning electron microscopy (SEM) images were taken with a Zeiss Supra 55VP microscope by using an acceleration voltage of 0.5 kV after depositing the samples onto porous membranes (0.1-μm-diameter pore size, Whatman) and coating them with carbon. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.