Nanowire transistor-based ultrasensitive virus detection with reversible surface functionalization.

We have applied a reusable silicon nanowire field-effect transistor (SiNW-FET) as a biosensor to conduct ultrasensitive detection of H5N2 avian influenza virus (AIV) in very dilute solution. The reversible surface functionalization of SiNW-FET was made possible using a disulfide linker. In the surface functionalization, 3-mercaptopropyltrimethoxysilane (MPTMS) was first modified on the SiNW-FET (referred to as MPTMS/SiNW-FET), with subsequent dithiothreitol washing to reduce any possible disulfide bonding between the thiol groups of MPTMS. Subsequently, receptor molecules could be immobilized on the MPTMS/SiNW-FET by the formation of a disulfide bond. The success of the reversible surface functionalization was verified with fluorescence examination and electrical measurements. A surface topograph of the SiNW-FET biosensor modified with a monoclonal antibody against H5N2 virus (referred to as mAb(H5)/SiNW-FET) after detecting approximately 10(-17) M H5N2 AIVs was scanned by atomic force microscopy to demonstrate that the SiNW-FET is capable of detecting very few H5N2 AIV particles.

Monitoring extracellular K+ flux with a valinomycin-coated silicon nanowire field-effect transistor.

A silicon nanowire field-effect transistor (SiNW-FET) coated with a polyvinyl chloride (PVC) membrane containing valinomycin (VAL) was employed as a biosensor (referred to as VAL-PVC/SiNW-FET) to detect the K(+)-efflux from live chromaffin cells. The detection sensitivity of K(+) with the VAL-PVC/SiNW-FET covers a broad range of concentrations from 10(-6) to 10(-2) M. The apparent association constants between VAL and Li(+), Na(+), K(+), and Cs(+) in Tris buffer solution were determined to be 67±42, 120±23, 5974±115, and 4121±140 M(-1), respectively. By culturing chromaffin cells on the VAL-PVC/SiNW-FET, the conductance was significantly increased by nicotine stimulation in a bath buffer without Na(+). The K(+) concentration at the cell surface was determined to be ~20 µM under the stimulation of 5 mM nicotine. These results demonstrate that the VAL-PVC/SiNW-FET is sensitive and selective to detect the released K(+) from cells and is suitable for applications in cellular recording investigations.

Surface potential variations on a silicon nanowire transistor in biomolecular modification and detection.

Using a silicon nanowire field-effect transistor (SiNW-FET) for biomolecule detections, we selected 3-(mercaptopropyl)trimethoxysilane (MPTMS), N-[6-(biotinamido)hexyl]-3(')-(2(')-pyridyldithio) propionamide (biotin-HPDP), and avidin, respectively, as the designated linker, receptor, and target molecules as a study model, where the biotin molecules were modified on the SiNW-FET to act as a receptor for avidin. We applied high-resolution scanning Kelvin probe force microscopy (KPFM) to detect the modified/bound biomolecules by measuring the induced change of the surface potential (ΔΦ(s)) on the SiNW-FET under ambient conditions. After biotin-immobilization and avidin-binding, the ΔΦ(s) on the SiNW-FET characterized by KPFM was demonstrated to correlate to the conductance change inside the SiNW-FET acquired in aqueous solution. The ΔΦ(s) values on the SiNW-FET caused by the same biotin-immobilization and avidin-binding were also measured from drain current versus gate voltage curves (I(d)-V(g)) in both aqueous condition and dried state. For comparison, we also study the ΔΦ(s) values on a Si wafer caused by the same biotin-immobilization and avidin-binding through KPFM and ζ potential measurements. This study has demonstrated that the surface potential measurement on a SiNW-FET by KPFM can be applied as a diagnostic tool that complements the electrical detection with a SiNW-FET sensor. Although the KPFM experiments were carried out under ambient conditions, the measured surface properties of a SiNW-FET are qualitatively valid compared with those obtained by other biosensory techniques performed in liquid environment.

Label-free detection of protein-protein interactions using a calmodulin-modified nanowire transistor.

In this study, we describe a highly sensitive and reusable silicon nanowire field-effect transistor for the detection of protein-protein interactions. This reusable device was made possible by the reversible association of glutathione S-transferase-tagged calmodulin with a glutathione modified transistor. The calmodulin-modified transistor exhibited selective electrical responses to Ca2+ (> or = 1 microM) and purified cardiac troponin I (approximately 7 nM); the change in conductivity displayed a linear dependence on the concentration of troponin I in a range from 10 nM to 1 microM. These results are consistent with the previously reported concentration range in which the dissociation constant for the troponin I-calmodulin complex was determined. The minimum concentration of Ca2+ required to activate calmodulin was determined to be 1 microM. We have also successfully demonstrated that the N-type Ca2+ channels, expressed by cultured 293T cells, can be recognized specifically by the calmodulin-modified nanowire transistor. This sensitive nanowire transistor can serve as a high-throughput biosensor and can also substitute for immunoprecipitation methods used in the identification of interacting proteins.

Analysis of the nucleoside triphosphatase, RNA triphosphatase, and unwinding activities of the helicase domain of dengue virus NS3 protein.

The helicase domain of dengue virus NS3 protein (DENV NS3H) contains RNA-stimulated nucleoside triphosphatase (NTPase), ATPase/helicase, and RNA 5'-triphosphatase (RTPase) activities that are essential for viral RNA replication and capping. Here, we show that DENV NS3H unwinds 3'-tailed duplex with an RNA but not a DNA loading strand, and the helicase activity is poorly processive. The substrate of the divalent cation-dependent RTPase activity is not restricted to viral RNA 5'-terminus, a protruding 5'-terminus made the RNA 5'-triphosphate readily accessible to DENV NS3H. DENV NS3H preferentially binds RNA to DNA, and the functional interaction with RNA is sensitive to ionic strength.