In situ detection of chromogranin a released from living neurons with a single-walled carbon-nanotube field-effect transistor.

Nanotubeor nanowire-based field-effect transistors (NT/ NW-FETs) as biosensors have recently drawn more and more attention in biological research because of their selectivity, sensitivity, and real-time detection capabilities. For biological studies, several one-dimensional semiconducting materials, such as silicon nanowires, carbon nanotubes, and indium oxide nanowires, have been applied as sensing elements to construct NT/NW-FETs for a variety of successful detections of proteins, nucleic acids, 8] cancer markers, and viruses. In particular, Lieber0s group recently applied silicon NW-FETs to monitor the electrical signals from single mammalian neurons, where each nanoscale nanowire–axon junction was used for spatially resolved, highly sensitive detection, stimulation, and inhibition of neuronal signal propagation. Their elegant, noninvasive measurements of the rate, amplitude, and shape of signals propagating along individual axons and dendrites by the use of silicon NW-FETs have opened a new avenue to the study of neurosciences. Chromogranin A (CgA), a protein with a calculated molecular weight of 50 kDa (apparent molecular weight 74– 80 kDa), is one of the molecules released from secretory vesicles when fused with the plasma membrane. CgA has many important biofunctions, for example, an innate immunity to fight against bacterial infection, as a mediator for neuron inflammation, and as a Ca chelator to modulate the secretion of other functional biomolecules and to mediate neuronal apoptosis. It has also been reported that the appearance of CgA in plasma is an excellent marker for the diagnosis of neuroendocrine tumors and neurodegenerative diseases, such as Parkinson0s and Alzheimer0s diseases. Therefore, the detection of CgA can be an indicator of synaptic activity when exocytosis occurs at the presynaptic terminal and a diagnostic tool for clinical examination. In this work, we chose single-walled carbon nanotubes (SWCNTs) to configure FETs on account of their high biocompatibility for neuron cells and their sensitive detection capability of the CgA released from living cells. The experimental results show that the SWCNT-FETs are a promising biosensor to monitor the in situ release of CgA during synaptic transmission among primary cultured embryonic cortical neurons, and can be used as a new tool to examine the synaptic activity. A schematic illustration of SWCNT-FET biosensors is shown in Figure 1. The FET devices were fabricated following a standard photolithographic procedure. After the fabrication of outer electrodes (represented in white in Figure 1a) with Au/Cr (50/50 nm in thickness), SWCNTs 2 nm in diameter (Thomas Swan & Co. Ltd.) were transferred to a SiO2/Si substrate (400-nm-thick SiO2) by dip-coating the substrate in a solution containing the SWCNTs and 0.125% sodium dodecylbenzenesulfonate (NaDDBS) surfactant following the method developed by Islam et al. After baking at 200 8C for 12 h to remove the NaDDBS, the as-dispersed SWCNTs in the central area (the reddish rectangles in Figure 1a,b) were electrically contacted by 50-nm-thick Cr leads (represented in yellow in Figure 1a), which were further passivated with a layer of SiO2 50 nm in thickness by thermal evaporation to avoid electric leakage to the sample solution. The original pattern design and photolithographic fabrication for this kind of device array by Lieber0s group can be found in References [10,11,20]. Ni/Au (10/50 nm in thickness) layers coated on the bottom of the SiO2/Si substrate were used as a back gate. The as-fabricated SWCNT-FETs were then converted to functional sensors by immobilizing the complementary probes, which will later be used against target molecules, onto the surfaces of SWCNTs (Figure 1c). In our experiment, a goat immunoglobulin G (IgG) antibody against CgA, denoted CgA-Ab (Santa Cruz Inc.), served as the probe and was immobilized on the sensing devices of the SWCNT-FETs with a literature-reported procedure developed by Dai and co-workers. After 1-pyrenebutanoic acid succinimidyl ester was applied as a linker to connect the antibody with the SWCNTs, Tween 20 was further used to block nonspecific binding. Figure 2a shows a representative scanning electron microscopy (SEM) image of a pair of source and drain elecACHTUNGTRENNUNGtrodes connected by a bundle of SWCNTs. The electrical transporting property of the SWCNT-FETwas characterized [*] C. -W. Wang, H. -C. Wu, Prof. Y. -T. Chen Department of Chemistry National Taiwan University Taipei 106 (Taiwan) Fax: (+886)2-2362-0200 E-mail: ytchen@pub.iams. sinica.edu. tw